Alkane monooxygenase study by molecular modelling techniques

Alkane monooxygenase study by molecular modelling techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Alkane monooxygenase study by molecular modelling techniques Elias Silva Santos, Elias Ramos Souza This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1486763/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Biodiesel biodegradation has been a limiting factor for its use as an alternative for petrol diesel. Thanks to low structural complexity, a group of enzymes such as alkane monooxygenase ( ladA ) can readily degrade several fatty acids, but the understanding of the mechanism of degradation is unclear yet. Then, we combined molecular docking and hybrid molecular dynamics (QM/MM) approaches for the study of ladA . The results showed that the bending of the isoalloxazine ring to the original plan enhanced its electrical coupling with a substrate. The flavin mononucleotide-FMN oxidation yields an activation energy of 74.29 kJ.mol -1 , which is necessary to make an oxygen molecule reactive. In addition, the initial activation energy determined for palmitic acid was 59.82 kJ.mol -1 and 64.64 kJ.mol -1 for linoleic acid. The diffusion of oxygen molecules into the active site is controlled by the movement of some residues that compose the tunnel access or by a water bridge. biodiesel petrol diesel biodegradation alkane monooxygenase simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Petroleum dependence as an energy source has become increasingly important, especially in emerging countries, which contribute to the acceleration of global climate change due to excessive emissions of particulate matter and greenhouse gases. This truth has led many nations to seek new alternatives for energy production, wherein biodiesel has emerged as one of the main renewable energy resources in its energetic matrix. According to Gucciardi [ 1 ], there is a growing tendency to exchange fossil fuels for biofuels. In pure form or, more commonly, in diverse proportions combined with traditional oil-derived diesel [ 2 ]. A few years ago, Brazil adopted the utilisation of blend biodiesel and diesel for economic and environmental purposes. The whole substitution of diesel by 100% vegetable oil is not applicable, because pure oil as a fuel has two severe problems: one is oil deterioration and the other is incomplete combustion in diesel engines [ 3 ]. Additionally, factors such as elevated temperature and the presence of light or extraneous materials such as metals or initiators [ 4 ] influence the self-oxidation and nature of reactions [ 5 – 6 ]. A relevant issue in the productive chain is the biodegradation of petrol diesel that has caused serious environmental and economic problems, mainly due to corrosion or blockage of pipelines in the storage. The biodiesel predominantly consists of eight different fatty acids methyl-esterified as oleate, palmitate, linoleate, laureate, and linoleic acid. The methyl esters present in biodiesel have a comparatively high octane number and are considered a biodegradable alternative to recalcitrant gasoline oxygenates [ 7 ]. Nevertheless, because fatty acids and aromatic compounds are of high relative molecular weight, some microorganisms have demonstrated the potential to degrade biodiesel more easily than petroleum diesel [ 8 ]. In some cases, metabolic activity can change the chemical sort of the compound (biotransformation) but not lead to complete mineralisation [ 9 ]. Biofuel contamination by microorganisms, especially in storage systems, primarily occurs in the presence of water in storage tanks [ 10 ]. The water absorbed by biodiesel through flushing enhances the oxidation and compromises the quality of the fuel stored, clogging of filters and nozzles due to the condensation reactions involving double bonds, which results in the formation of gums and sediments, increasing the viscosity of biodiesel [ 11 ]. Microbes activate alkanes under aerobic or anaerobic conditions with different enzyme systems [ 12 ]. Under aerobic conditions, oxygen serves as the electron acceptor whereas under anaerobic conditions; sulphate and nitrite accept electrons to complete the processes [ 12 ]. The whole reaction catalysed by flavoprotein monooxygenase involves three chemical processes: a) NAD(P)H reduction of flavin; b) reaction of the reduced flavin with O 2 to produce a C 4a -flavin [hydro] peroxide; and c) binding, orienting and activating the substrate for oxygenation [ 13 ]. Furthermore, the mechanism of transferring labile reduced flavin from the reductase to the oxygenase is unknown [ 13 ] and the reduced enzyme-bound flavin reacts with oxygen to form C 4a – (hydro) peroxyflavin, which is stable in the absence of substrates [ 13 ]. The aerobic biodegradation of a hydrocarbon is a process that depends on several factors, from the number of hydrocarbons to the quantities of oxygen molecules present in the environment [ 14 ]. In many ecosystems, there is already an adequate indigenous microbial community capable of extensive oil biodegradation, if environmental conditions are favourable for oil-degrading metabolic activity [ 15 ]. A group of microorganism enzymes that use carbon as an energy source mediates the nucleophilic attack of the aliphatic chain or aromatic parts of hydrocarbon culminating in the production of primary alcohols [ 12 ]. In this approach, alkanes are first attacked at their terminal methyl group to yield the corresponding primary alcohols, which are further oxidised by alcohol and aldehyde dehydrogenases [ 16 ]. The terminal oxidation pathway undergoes ω-hydroxylation at the terminal methyl group (the ω position); yielding ω-hydroxy fatty acid that is further converted to a dicarboxylic acid, which then also enters β-oxidation [ 16 – 18 ]. Additionally, it has been suggested that an oxygen-independent formation of a terminal double bond occurs as an alternative mechanism during aerobic growth on alkanes [ 19 ]. The three-dimensional structures of long-chain alkane hydroxylases remained unclear until the 2.7 Å apoenzyme and 1.9 Å holoenzyme structures of ladA , a long-chain alkane monooxygenase to be reported in 2008 [ 20 ]. The ladA was isolated from the thermophilic bacillus Geobacillus thermodenitrificans NG80-2, which utilises terminal oxidation as a pathway for the conversion of long-chain n -alkanes. Alkane monooxygenase is one of three enzymes involved in the process of aerobic biodegradation. It is a homodimer composed of (β/α8) barrel fold capable of degrading alkanes for the conversion of long-chain n-alkanes from C 15 to at least C 36 by introducing oxygen into the substrate [ 20 ]. The structure of ladA contains a triose phosphate isomerase (TIM) barrel fold that differs from the prototypical TIM barrel structure due to five extended insertion regions and an extension at the C-terminus of the polypeptide chain [ 20 ]. A pocket at the C-terminal entrance of the TIM barrel is sufficiently large to accommodate FMN, O 2 , and part of the terminal of a long-chain n -alkane. The flavin ring of FMN is located in the barrel and its S i -face is exposed to the solvent [ 20 ]. The ribityl side chain and phosphate moieties are inserted between strands β4 and β5 of ladA [ 20 ]. A cavity above the Si -face of FMN consists of residues Phe10, Met12, His17, Ala57, Val59, Tyr63, Gln79, His138, His311, Trp348, and Phe349 with polar residues concentrated on the left sphere and hydrophobic residues on the right sphere [ 20 ]. When supplied by different enzyme systems, through the reaction of the reduced flavin with O 2 , ladA provides C 4a -flavin (hydro) peroxide activating the substrate [ 20 ]. This enzyme creates a highly potent oxidizing agent from oxygen in the active site, which overcomes the low reactivity of the hydrocarbons and allows the introduction of functional groups that enable further biochemical conversions [ 21 ]. Alkane hydroxylases are widespread in petroleum-degrading bacteria and a number of them are efficient at oxidising substrates [ 12 ]. Hence, there is considerable interest in employing alkane hydroxylases for industrial applications. Thermophilic long-chain n -alkane-degrading bacterial strains are of particular interest for their biotechnological applications [ 12 ]. Although many of the Geobacillus come from high-temperature environments, it has been pointed out that they are frequent in temperate-climate soil environments where they are unable to grow due to temperature restrictions [ 22 , 23 ]. In addition, there are several advantages to using thermophilic microorganisms in the bioremediation of hydrocarbons over mesophilic organisms. For instance, the production of biosurfactants that act as solubilising agents. As such, ladA is a perfect target for study, especially as an alternative to recovery areas affected by oil spillage. So far, few alkane hydrolases representatives of long-chain have been characterised. Then, this work has the goal of supplying new insight into the dynamics and structural changes of ladA extracted from Geobacillus thermodenitrificans NG80-2, by introducing two aliphatic compounds of biodiesel as substrates and O 2 molecules for more explanation of the ladA structure and the effects of dissolved oxygen in a salt environment. Here, a hybrid quantum mechanics/molecular mechanics (QM/MM) approach simulation is applied for the investigation of the catalytic mechanism of oxidation involving FMN and substrate interaction. A computational estimate of the binding free energy was also presented to elucidate the interaction formed between the cofactor FMN and the substrate. 2 Materials And Methods 2.1 Molecular docking 2.1.1 Preparation of the receptor and ligands The structure of ladA obtained from the Protein Data Bank (PDB CODE 3B9O) is a target for study. Autodock Tools [ 24 ] was used to create a model of the A chain by introducing hydrogen and its protonation states were optimised. Three structures of linoleic acid, COD-CID 5280450, palmitic acid COD-CID 985, and FMN COD-CID 643976 were obtained from the PubChem database (pubchem.ncbi.nlm.nih.gov/) in the SDF file format. We used OpenBabel software version 2.3.1 [ 25 ] to convert them into PDB format. All bonds were defined to be rotational and bonded using the Autodock Tools for protonation states and BCC method implemented on ACPYPE [ 26 ] calculated partial charges. The binding site selected for docking is formed by including a group of residues based on knowledge of the amino acids that constitute the active site, such as His17, Met12, Ala57, Val59, Tyr63, Gln79, His311, His138, and FMN. We deleted all structural waters except Wat528 and Wat575 because the presence of these water molecules plays an important role in the accuracy of ligand-protein docking predictions in this system. 2.2 Redocking Redocking simulation was performed by removing the FMN from the ladA structure and reconnecting it to the protein to adjust the docking parameters using AutoDock Vina [ 27 ]. The box simulation was defined around the coordinates of the FMN centre (-26.391, 35.045, 19.412) with a size of 25 x 25 x 25 Å 3 and a grid spacing of 0.375 Å was established. Table 1 summarises the best parameters achieved with such a procedure. The ‘energy range’ describes the maximum energy difference between the best binding mode and the worst one displayed (given in kcal/mol); ‘num modes’ parameter corresponds to the maximum number of generated binding modes. The ‘exhaustiveness’ associated with the thoroughness of the search is roughly proportional to the time and ‘seed’ of the random number generator. The most accurate result was compared with the FMN crystallography position as deduced from the superposition of the two structures (Fig. 1 ). The evaluated RMSD value (2.04Å) ensures that molecular docking represents a good conformation. Table 1 Parameters used to set docking Parameter Value Energy range 10 Num modes 20 Exhaustiveness 150 Seed -5594 2.3 Protein-ligand docking AutoDock Vina accomplished the docking of linoleic acid and palmitic acid into ladA . The best binding mode procedure obeyed two steps: identical solutions in terms of conformation were separated and results with lower energy allocated in families. The selection of complex used as a template for molecular dynamics was based on the hydrogen bond number between FMN and substrate among those with lower binding energy. The complexes were visualised using VMD 1.9.1 [ 28 ] and submitted to optimisation using the Steepest Descent algorithm implemented in GROMACS 5.1.2 [ 29 ]. 2.4 Classical molecular simulation For the models obtained from the docking procedure, classical molecular dynamics simulation was performed using the Charmm27 force field [ 30 ] implemented in the GROMACS package. The topologies of linoleic acid, palmitic acid, and FMN were obtained from the server Web SwissParam [ 31 ] and partial charges were assigned through ACPYPE. The LINCS algorithm [ 32 ] applied to all bonds in the complex allowed 1 fs time steps and the SETTLE algorithm [ 33 ] for water molecule bonds. The cut-off of 1.1 nm was used to calculate non-bonded interactions, resorting to the PME method [ 34 ]. The V-rescale method [ 35 ] coupled with a time constant of 1 ps kept the temperature at 300 K, while the Parrinello-Rahman method [ 36 ] with 5 ps time constant relaxation to remain the pressure of 1 bar. For this study three systems were built: The primary system, named LAD_L (with linoleic acid) was soaked in a box of volume 4356.49 nm 3 with ~ 17000 water molecules of TIP3P type and 34 oxygen molecules distributed randomly around the protein surface. We made the O 2 concentration low to make sure it does unsaturate in water at ambient conditions, warranting enough samples of O 2 to the pathway in the protein. The margin of 1.2 nm between protein and every side of the box as well as for the second system labelled LAD_P (with palmitic acid). The third system, called LAD_F was made equivalently, but without substrate or oxygen molecules. NaCl molecules at 0.15 M concentration neutralise the systems by equilibrating them during 40,000 ps with a restriction position for protein. Then, we ran simulations for each system for 200,000 ps, while the Maxwell–Boltzmann distribution generated initial velocity and Xmgrace version 5.1.22 built graphs. For obtaining an insight into the binding energy between FMN and the substrate, we performed a calculation based on Poisson-Boltzmann surface area (MM-PBSA) [ 37 ]. This method combines three energy terms to explain the change in the binding free energy according to the equation: ΔG bind = ⟨G PL ⟩ - ⟨G P ⟩ - ⟨G L ⟩, (1) G PL , G P and G L are the total free energy of the protein-ligand complex, the total free energies of the isolated protein, and the ligand in a solvent, respectively. Eq. (1) is estimated from ΔG bind =E bond + E ele + E vdw + G pol + G np -TΔS, (2) The first three terms are the MM energy terms from bonded i.e., bond, angle, and dihedral. The third and fourth terms refer to electrostatic and van der Waals interactions. G pol and G np are the polar and non-polar contributions to the solvation free energies, and TΔS is the entropy term. For configurational entropy calculation, we used the Schlitter method [ 38 ]. 2.5 Quantum mechanics (Reaction path calculation) Two steps were considered in the oxidation reaction: the first step is the reaction of O 2 with reduced FMN, and the second one is the reaction of the O atom with the substrate. We performed the activation energy calculations with the nudged elastic band (NEB) method [ 39 ], implemented in Quantum Espresso suite version 6.0 (QE) [ 40 ]. NEB creates a set of images of the system connected with springs to make a representation of a configuration path. In estimating activation energy, the representative initial structure selected at the latter time of the trajectory was obtained from GROMACS. Due to computational limitations, executed calculations in the subsystem containing the substrate, FMN and O 2 because of the expensive computations. Reactant geometries were estimated by optimising the geometry with the conjugate gradient algorithm implemented on QE. The climbing image method was used for the highest energy image, increasing the accuracy of the calculations [ 41 , 42 ] and fixed all atom positions in the system except for the atoms of the O 2 molecule. In addition, we considered for both systems the following reaction coordinates r = d (C 4a -O - CH/substrate) as shown in the Table (Online Resource 1). 3 Results And Discussion 3.1 Docking simulations The characterisation of ladA and substrates through docking simulation shows the ways of bonding the substrates attached to the cavity of the hydrophobic ladA (Figs. 2 a and 2 b). The results were analysed based on the lowest binding energy values. The findings have shown that hydrogen bond plays a critical role in the stabilisation of the substrates in both complexes, but some hydrophobic contacts and salt bridges help to equilibrate them into the active site. In addition, the water molecules (Wat528 and Wat575) are involved in protein-substrate recognition mediating hydrogen bonds between the protein and the substrate. Linoleic acid is perfectly bound within the active region by hydrogen bonds, which produces energy of -0.73 kcal.mol − 1 (-3.05 kJ.mol − 1 ). Added to that, hydrophobic contacts with Ile18, Thr316, Trp348, Phe349, and salt bridge help counteraction it. Likewise, palmitic acid maintained steady to the active site thanks to the energy of -0.65 kcal.mol − 1 (-2.72 kJ.mol − 1 ) provided by hydrogen bonds. Hydrophobic contacts with Trp348, Phe349, and bridge salt collaborate with the balance. Nevertheless, the data show that the total electrostatic interaction has a smaller influence on the stabilisation of substrates, i.e. -0.035 kcal.mol − 1 (-0.14 kJ.mol − 1 ) for the linoleic acid and 0.043 kcal.mol − 1 (0.18 kJ.mol − 1 ) palmitic acid. The H 5 atom bonded to the N 5 atom of the isoalloxazine ring directly points to the H atom of the carboxyl group of linoleic acid, the binding energy of -5.52 kcal.mol − 1 (-23.10 kJ.mol − 1 ) (average distance 2.3 nm) and − 5.41 kcal.mol − 1 (-22.64 kJ.mol − 1 ) for palmitic acid whose average distance is 2.7 nm. Van der Waals interaction − 8.54 kcal.mol − 1 (-35.73 kJ.mol − 1 ) (linoleic acid) and − 7.81 kcal.mol − 1 (-32.68 kJ.mol − 1 ) (palmitic acid) is responsible for maintaining the two substrates firm in the complexes. In summary, the docking results show the significance of van der Waals interaction for the stabilisation of the substrates within the active region of ladA . 3.2 Molecular dynamics 3.2.1 Overall structure After identifying the best complex from molecular docking, molecular dynamics simulation was used to estimate the root-mean-square deviation (RMSD) of the protein for the quantification of the degree of conformational changes during MD simulations (Fig. 3 a). The analysis of protein stability was fulfilled inspecting the conformational states takes into account the changes in the position of atoms concerning the optimised structure, which revealed that LAD_L and LAD_P have relative stability around 0.22 nm and 0.30 nm, respectively, as can be seen through some plateaus in their trajectories. Alternatively, the LAD_F plot presented the highest increase in RMSD during the last 100,000 ps of the simulation, which shows many conformational changes as a reflection of local fluctuations in some properties of the protein, such as shape and size. Therefore, the stability of ladA is altered when substrates bind, inducing changes in structure and conformational flexibility. We also performed the principal component analysis (PCA), where the set eigenvectors were obtained from backbone trajectories during the same period of simulation used to calculate the RMSD through the projection of two principal components (Fig. 3 b). The data show higher dispersing in LAD_P confirming the great flexibility of the loop regions exposed to the solvent, wherever one of these loops works as a lid for a tunnel of access to the active site as shown in Fig. 3 c. Additionally, the loop in the active site region constituted by residues Ala57, Asp58, Val59, Val60, and Gly61 undergoes different conformational arrangements induced by substrates. For example, the stretched conformation and orientation in the last picoseconds of simulation in LAD_P increases the contact with Asp348. In contrast, the orientation of the linoleic acid reduces its contact with Asp348. As such, the substrate depends on the conformational changes in the loop to be catalysed. The RDF-radial distribution function represented by g(r) in Fig. 4 displays the distribution of water around the centre of mass of the FMN. The first hydration shell consists of water at 0.18 nm and the second one occurs at 0.21 nm. The movement of water to greater distances from the FMN caused by substrates gradually assumes the bulk of the water from this region. Hydrogen bonds formed with amino acids from the active site plus water molecules around the isoalloxazine ring and ribityl group, water bridges, and hydrophobic contacts maintain FMN tight in the packed hydrophobic core. This result suggests the formation of a hydrophobic environment surrounding FMN and the three water found there should play an important role in the catalytic process. As shown in the Figure (Online Resource 2), the number of these contributions is a limiting factor for conformational changes or loss of vibrational entropy thanks to the constraints of binding. 3.2.2 Diffusion of O 2 molecule into ladA Diffusion and reaction conversion of O 2 requires the initial concentration for substrate biodegradation. Many enzymes, such as Cytochrome cOxydases [ 43 ] use molecular oxygen to explore tunnels for controlling specific substrates or transport ions from the surface of the protein to the active site, acting as pathways for substances between the bulk solvent and the active site. The ladA has many tunnels to transport oxygen molecules, considering that oxygen molecule is transient at the active site, but few are used effectively. This section presents results obtained from the analysis of possible tunnels for oxygen molecule transport through ladA , utilising the MOLE Online web server [ 44 ]. The O 2 explores some cracks on the surface of the protein to find cavities formed mainly by hydrophobic residues. The results insinuate the existence of many tunnels, but four are probable for transport because of their low curvature and short length (Fig. 5 a). In analysing these tunnels, the T1C1 tunnel connected to the T2C1 is lined by Tyr66, Arg67, Ser69, Asp71, Thr72, Arg75, Ala310, Tyr63, Val65, and His318, Gly314, Gly317, respectively. However, they are directed away from the active site. T3C1 tunnel covered by residues His311, Tyr312, Thr316, Trp348, Phe349, Gly347, and Gly315 is wide enough to allow the substrate to reach the catalytic centre. Nevertheless, as oxygen is a hydrophobic molecule, it must prefer hydrophobic channels where it can use the dynamics structure to reach the active site spreading through T4C1 coated by residues Val59, Val60, Asp58, Asp84, Phe105, Ser137, Ser106 and length of 16.5Å (Fig. 5 b). Therefore, these data reveal that oxygen molecules and the substrate use distinct routes for reaching the reactive centre. For alternative tunnels, they seem unlikely because longer side-chains can obstruct the passage of O 2 due to conformational changes or are not hydrophobic routes. In Lad_L, the first oxygen enters the active site at 6,311 ps and remains there for a long time. However, in Lad_P the first oxygen reaches the active site cavity at half time of simulation, although many more oxygen molecules come to the active site in both systems. Translational diffusion of oxygen molecule into the active site was computed and shown in Table 2 at the interval of 100,000–150,000 ps. According to data, the largest flux of oxygen molecules is found to be in the x-direction (LAD_P) and y-direction (LAD_L). This behaviour seems to be due to the substrate length and conformational changes of the residues around the substrate. The diffusion decreases with obstructions caused by solidary movement of some residues from the tunnel or by water bridge formation as a response to substrate conformational changes. Hence, the amino acids that compose the tunnel play an important role in the diffusivity of the oxygen molecule and, consequently, the capacity of the enzyme to degrade substrates. According to Fig. 6 , the fluctuations of the protein residues (RMSF) in the systems were virtually similar and certain residues of the active site have highly fluctuated. Comparing the RMSFs for the binding site of systems, we can see that, on average, the amino acids bind the ligand and keep them firm throughout the simulation. In LAD_P, Tyr63 shows more fluctuation than LAD_L or LAP_F, but surprisingly, His311 and His138 fluctuated less in the bounded systems than in the unbounded. Additionally, when the histidines in bounded systems are compared, the fluctuation is major in LAD_P, suggesting that the binding of a certain substrate cannot stabilise the complex. Thereby, speculation by Liu et al. [ 20 ] that the substrate-binding mode would require substrate specificity and determine the range of alkane chain length for catalysis finds support. Carbon-chain lengths of C 14 and lower would be so short that the terminal carbon could not reach the active site while the opposing terminal part was anchored to the protein [ 20 ]. In contrast, carbon-chain lengths of C 15 and higher would bind with the exact carbon chain fitting from the active site to the anchoring site. Indeed, the findings of Owsianiak et al. [ 45 ] using a consortium of bacteria isolated from an oil-contaminated site showed complete degradation of methyl-esters in blends of biodiesel fuels, and more than 50% C 16 (palmitic acid) was degraded. In the same way, Miller and Mudge [ 46 ] observed that unsaturated (C 18 ) fatty acids were degraded more rapidly than the methyl-esters of saturated fatty acids. Therefore, biodegradability has to be a function of chain length, conformation, degree of substitution (unsaturation), C = C position, and cis and trans configurations [ 47 – 49 ]. Table 2 The oxygen diffusion coefficient in different directions (x10 − 2 cm 2 .s − 1 ) LAD_P LAD_L D x 0.88 0.47 D y 0.86 0.73 D z 0.58 0.67 *The standard error of 0 is not shown 3.2.3 FMN binding Changes in the angles formed between the atoms (C 4a -N 10 -C 10 ) named as plane 1 and (C 9A -C 5A -C 10 ) as plane 2 and the distance between (N 10 -C 4' ) atoms of the isoalloxazine ring were monitored and summarised in Table 3 . In terms of classical molecular dynamics, the bending motion of the isoalloxazine ring is practically the same for all systems. Some residues Gly315, His154, Asp155, and Ser230 interact with FMN tail making hydrogen bonds that restrict the movement of tail keeping the average distances between N 10 -C 4' around 0.18nm (Lad_P and Lad_F) and 0.23 and Lad_L. However, as shown below, an examination of the ab initio results in both bonded systems revealed that the structure of the isoalloxazine ring displayed some distortion to your original plan, which is not revealed by classical simulations because the force field is not capable to show this distortion, caused by the electronic change in the isoalloxazine ring. The results indicate plane 1 rotates to 121.91 ◦ (LAD_P) and 90.75 ◦ (LAD_L) while plane 2, 93.80 ◦ (LAD_P), and 77.95 ◦ (LAD_L) in favour of substrate cleavage. As such, changes in the isoalloxazine ring modulate the electron transfer process producing different orientations and distances between the reactive centre and the substrate, improving the electrostatic interaction between them. According to the proposed mechanism by Liu et al. [ 20 ], His138 must act as a proton donor in the reaction between reduced flavin and an oxygen molecule that leads to the formation of the C 4a -hydroperoxyflavin intermediate. Direct proton transfer requires the proximity of the proton donor and acceptor to be within the distance of a typical H-bond (∼0.28 nm between heavy atoms) [ 50 ]. Here, and for geometric reasons, His138 cannot directly transfer a proton to the isoalloxazine ring (0.35–0.69 nm distance) unless the presence of water molecules near the isoalloxazine ring is imperative. According to the data obtained in these simulations, His138 helps to steady the ribityl group and the phosphate group with one water bridge in LAD_P (please, see again Online Resource 2). Additionally, the same data suggest that transfer could occur among isoalloxazine ring, one water and Thr104 in LAD_L or by a hydrogen bond formed with Asp58 in LAD_P owing to specific changes in the protein structure produced by water reorientation or polar side chain positions in the active site [ 51 ]. That reorientation must change the protonation state during the reaction. In bounded systems, the substrate has some conformational mobility during the simulation coming to the isoalloxazine ring. This approximation displaces the water close to the isoalloxazine ring, creating a hydrophobic microenvironment between them. Linoleic acid is stabilised by Asp58 and Lys347, which can be involved in substrate activation during hydroxylation, and Trp348 steady palmitic acid. Table 3 FMN average angles and distances isoalloxazine ring Angle (deg) LAD_P LAD_L LAD_F Plane 1 29.32 (± 0.01) 29.38 (± 0.01) 29.27 (± 0.01) Plane 2 89.11 (± 0.01) 89.17 (± 0.01) 88.99 (± 0.01) Distance (nm) 0.18 (± 0.01) 0.23 (± 0.01) 0.18 (± 0.01) *Standard errors are given in parentheses 3.2.4 FMN interaction with the substrate In this section, the binding free energy change is computed through the MM-PBSA approach for FMN-palmitic acid and FMN-linoleic acid, taking into consideration configurational changes. Although methods to estimate the binding free energy, such as Free Energy Perturbation (FEP) or Thermodynamic Integration (TI) have more accuracy they are computationally expensive, thus we used MM-PBSA approach for calculation. The binding free energy was estimated by calculating the configurational entropy of the systems, where the dielectric constant (ɛ = 4) was assumed as the default to represent the internal dielectric constant. GROMACS tool computed the external dielectric constant whose value found was ɛ =101.07 and Schlitter method computed the configurational entropy. The configurational entropy found was 7534.66 J. (mol. K) −1 in Lad_L and 1363.93 J. (mol. K) −1 in Lad_P, where the loss entropic effect is smaller in Lad_P than Lad_L. The binding free energies between FMN-Palmitic acid and FMN-linoleic acid were estimated at -15.77 kJ.mol − 1 and − 12.13 kJ.mol − 1 , respectively. These values are different from those obtained by docking refining the results of the scoring function. Van der Waals energies of -45.53 kJ.mol − 1 (LAD_P) and − 64.09 kJ.mol − 1 (LAD_L) were critical contributions for binding free energies. The MM-PBSA approach must provide results of intermediate quality comparatively to the methods aforementioned (FEP and TI) for calculating binding free energy change, but it gives an idea of the binding energy between FMN and the substrates here studied, improving the results obtained through molecular docking simulations. 3.3 Studies of quantum mechanics In this moment of the study, an approach using quantum mechanics calculation was employed to investigate the oxidation mechanism for substrate mediated by oxidation of the FMN. Kinetic and chemical analysis suggest an initial electron transfer from singlet reduced flavin towards triplet O 2 to make a caged radical pair [ 52 ]. After spin inversion, the intermediate generated can collapse into a peroxyflavin that is unstable in water and heterolytically dissociates into H 2 O 2 and oxidised flavin [ 53 ]. Based on that reaction scheme, we took the last frame of GROMACS simulation to compute path reaction. The results show the first step of the bond formed between O 2 and C 4a of the isoalloxazine ring has an activation energy of 0.76 eV (74.29 kJ.mol − 1 ), i.e., the amount of energy required the reaction to occur. This energy reveals a high cost for translationing the O 2 towards the C 4a atom of the isoalloxazine ring, where two local minima and a saddle can be seen in the graph in the Online Resource 3. In addition, an experiment performed by Hasting and Gibson indicates that a reaction involving the oxidation of reduced flavin yields activation energy in appearance luminescence is about 0.56 eV (54.4 kJ.mol − 1 ) [ 54 ]. In the second step of the process, the oxygen atom activates palmitic acid with an energy of 0.61 eV (59.82 kJ.mol − 1 ) and linoleic acid 0.66 eV (64.64 kJ.mol − 1 ). The movement of the oxygen atom towards the substrate is shown in the animations ESM_1 and ESM_2 in the Online Resource, respectively, which help to clarify the pathway of the reaction. These findings were compared with the findings of Adachi et al., who estimated the activation energy of 50.1 ± 4.2 kJ.mol − 1 in a study with methylated linoleic acid in Autoxidation of Polyunsaturated Fatty acids [ 55 ]. Ishido et al. investigating the oxidation of linoleic acid, in the presence of saturated acids or its methyl esters, found an activation energy of 54.4 kJ.mol − 1 [ 56 ]. In another investigation conducted by Fu et al., the palmitic acid-deoxygenating at a high temperature found 79 ± 5 kJ.mol − 1 of activation energy [ 57 ]. Furthermore, Hermida et al. reported an activation energy of 111.57 kJ.mol − 1 for palmitic acid when using nickel as a catalyst for palmitic acid deoxygenation to synthesise n-pentadecane and 1-pentadecene [ 58 ]. Altogether, we consider that data obtained here are in relative concordance with experimental data, even not taking into account all the complexity of the active site. Although the activation energies calculations using SCF algorithms are well-known to have difficulties when attempting to enforce specific electron distributions [ 59 ], and therefore, encountering convergence difficulties may not point to an intrinsic instability of those pairs, but simply be a consequence of numerical/algorithmic difficulties [ 59 ]. Energetic minimisation performed in both systems respecting the reactant state using ab initio dynamics relaxed the bonds ( Fig. 7 ) with the coupling of the oxygen atom between carbons in the acids allowing movement of atoms of acids. In LAD_P, the distance increased from 0.18 nm to 0.27 nm, while in LAD_L increased from 0.17 nm to 0.25 nm. 4 Conclusions In this research, it has been possible to elucidate some dynamic and structural aspects of ladA . We investigated some characteristics of the mechanism of ladA degradation through the calculation of QM/MM. The results indicate that water provides a trail for charge transfer for FMN via hydrogen bonds. The reaction paths of oxygen molecules produce new information about the oxidation of FMN and substrates, where the data is close to the experimental findings, although it was not considered certain amino acids or water in the calculation of activation energies. New conformations of these residues or the presence of water molecules in the calculation must yield energy values that would explain the divergence with the experimental results, besides the limitations of the algorithms used. Indeed, the findings show that the enzyme controls the entrance of oxygen or ligands through conformational changes of the residues that form access tunnels from the enzyme surface, providing a favourable environment for the degradation of the selected substrates. This specific result also suggests that the availability of oxygen molecules in the environment determines the rate of degradation of fatty acid molecules that must be similar to those obtained experimentally. Therefore, this investigation provides new insight into the enzyme ladA as a good source for developing novel ways to improve production techniques and use for biodiesel that is still increasing in Brazil, representing in 2019 about 9.6% by the energy of diesel consumption, according to IEA World Energy Balance [ 60 ]. On the other hand, the use of this enzyme can mitigate the effect of pollution and recovery production areas degraded, including a reduction in the soil of toxic residue and improvement in production systems. Declarations Funding The authors declare that no funds, grants, or other supports were received during the preparation of this manuscript Competing interests The authors have no relevant financial or non-financial interests to disclose Author Contributions Elias Silva dos Santos contributed to the conceptualization, formal analysis, methodology, investigation, writing-review, editing and visualization Elias Ramos de Souza contributed to the review and editing Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request References Garcez Gucciardi CA, de Souza Vianna JN (2009) Brazilian Biodiesel Policy: Social and environmental considerations of sustainability. Energy 34:645–654 Ginn TR, Hatch TJ, McKone TE, Rice DW (2009) California Biodiesel Multimedia Evaluation Tier I Report. University of California, Davis and University of California, Berkeley Peterson CL, Auld DL, Korus RA (1983) Winter rape oil fuel for diesel engines: Recovery and utilization. JAOCS 60:1579–1587 Thomas AO, Leahy MC, Smith JWN, Spence MJ (2017) Natural attenuation of fatty acid methyl esters (FAME) in soil and groundwater. Q. J. Eng. Geol. Hydrogeol. 2016–2130 Knothe G (2007) Some aspects of biodiesel oxidative stability. Fuel Process Technol 88:669–677 Salam DA, Suidan MT, Venosa AD (2012) Effect of butylated hydroxytoluene (BHT) on the aerobic biodegradation of a model vegetable oil in aquatic media. ES&T46: 6798–6805 Liu S, Sulfita M (1994) Anaerobic biodegradation of methyl esters by Acetobacterium woodii and Eubacterioum limosum. J Ind Microbiol 13:321–327 Follis PA (1994) An Investigation into the biodegradability of soy diesel under various environmental conditions. Master’s Thesis. School of Natural and Environmental Sciences, Dusquesne University, 58 Atlas RM, Bartha R (1992) Microbial Ecology-Fundamentals and Applications 3ed. Benjamin Cummings, Redwood, p 563 Hannon M, Gimpel J, Tran M, Rasala B et al (2010) Biofuels from algae: challenges and potential. Biofuels 1(5):763–784 Lin C-Y, Chiu C-C (2009) Effects of Oxidation during Long-term Storage on the Fuel Properties of Palm Oil-based Biodiesel. Energy Fuels 23(6):3285–3289 Ji Y, Mao G, Wang Y, Bartlam M (2013) Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases. Front Microbiol 4:1–13 Ballou DP, Entsch B, Cole LJ (2005) Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases. 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BMC Res Notes 5:367 Trott O, Olson OJ (2010) Autodock Vina: Improving speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comp Chem 31:455–461 Humphrey W, Dalke A, Schulten K (1996) VMD –Visual Molecular Dynamics Molec. Graphics 141:33–38 Abraham MJ, van der Spoel D, Lindahl E, Hess B (2016) the GROMACS development team, GROMACS User Manual version 5.1.2, Foloppe N, MacKerell AD Jr (2000) All-Atom Empirical Force for Nucleic Acids: I) Parameter Optimization Based on Small Molecule and Condensed Phase Macromolecular Target Data. J Comp Chem 21:86–104 Zoete V, Cuendet MA, Grosdidier A, Michielin O (2011) SwissParam, a Fast Force Field Generation Tool for Small Organic Molecules. J Comput Chem 32:2359–2368 Ryckaert JP, Ciccotti G, Berendsen HJC (1977) Numeral integration of the Cartesian equation of motion of a system with constraints; molecular dynamics of n-alkanes. 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World Scientific, Singapore, p 385 Giannozzi P, Baroni S, Bonini N, Calandra M, Car R et al (2009) J. QUANTUM ESPRESSO: a modular and open-source software project for quantum simulations of materials Phys. Condens. Matter 21, number 21 395502 Henkelman G, Jónsson H (2000) Improved tangent estimate in the nudged elastic band method for finding minimum energy paths and saddle points. J Chem Phys 113:9978–9985 Henkelman G, Uberuaga BP, Jónsson H (2000) A climbing image nudged elastic band method for finding saddle points and minimum energy paths. J Chem Phys 113:9901–9904 Oliveira ASF, Damas JM, Baptista AM, Soares CM (2014) Exploring O2 Diffusion in A-Type Cytochrome c Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site. PLoS Comput Biol 10(12):e1004010. doi: 10.1371/journal.pcbi.1004010 Pravda L, Sehnal D, Toušek D, Navrátilová V, Bazgier V et al (2018) MOLEonline: a web-based tool for analyzing channels, tunnels and pores Nucleic Acids Res, ASAP, 46:W368–W373doi: 10.1093/nar/gky309 Owsianiak M, Chrzanowski L, Szulc A, Staniewski J, Olszanowski A et al (2009) Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: Effect of the type of blend and the addition of biosurfactants. Bioresour Technol 100:1497–1500 Miller NJ, Mudge SM (1997) The effect of biodiesel on the rate of removal and weathering characteristics of crude oil within artificial sand columns. Spill Sci Technol Bull 4:17–33 Maier R (2000) Microorganisms and organic pollutants (Chap. 16). In: Maier R, Pepper IL, Gerba, CP (Eds.). Environmental microbiology 363–401 Demirbas A (2009) Progress and recent trends in biodiesel fuels. Energy Convers Manag. doi: 10.1016/j.enconman.2008.09.001 Hill E, Hill GC (2009) Strategies for resolving problems caused by microbial growth in terminal and retail sites handling biodiesel. In: Proceedings of the 11th international conference on the stability, handling and use of liquid fuels, Prague, Czech Republic, 1–22 Migliore A, Polizzi NF, Therien MJ, Beratan DN (2014) Biochemistry and theory of proton-coupled electron transfer. Chem Rev 114:3381–3465 Neutze R, Pebay-Peyroula E, Edman K, Royant A, Navarro J, Landau EM (2002) Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport. Biochim Biophys Acta 1565:144–167 Romero E, Castellanos GJR, Gadda G, Fraaije MW, Mattevi A (2018) Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes. Chem Rev 118:1742–1769 Kemal C, Chan TW, Bruice TC (1977) Reaction of 3 O 2 with Dihydroflavins. 1. N 3, 5 -Dimethyl-1, 5-Dihydrolumiflavin and 1, 5-Dihydroisoalloxazines. J Am Chem Soc 99:7272–7286 Hastings JW, Gibson QH (1963) Intermediate in Bioluminescent Oxidation of Reduced Flavin Mononucleotide. J Biol Chem 238:2537–2554 Adachi S, Oshiguro T, Matsuno R (1995) Autoxidation Kinetics for Fatty Acids and Their Esters. JAOCS 72:547–551 Ishido E, Minemoto Y, Adachi S, Matsuno R (2001) Oxidation of Linoleic Acid and Methyl Linoleate Mixed with Saturated Fatty Acid or its Methyl Ester. U J Food Sci Technol 34:234–238 Fu J, Lu X, Savsge PE (2010) Catalytic hydrothermal deoxygenation of palmitic acid. Energy Environ Sci 3:311–317 Hermida L, Amani H, Abidullah AZ, Mohamed AR (2016) Deoxygenation of Palmitic Acid to Produce Diesel-like Hydrocarbons over Nickel Incorporated Cellular Foam Catalyst: A Kinetic Study. J Adv Chem Eng. doi: 10.4172/2090-4568.1000144 Silva PJ (2016) Refining the reaction mechanism of O 2 towards its co-substrate in cofactor-free dioxygenases. PeerJ. doi: 10.7717/peerj.2805 IEA Bioenergy (2021) Implementation of Bioenergy in Brazil. https://www.iea.org/policies?country=Brazil . Accessed 18 February 2022 Additional Declarations No competing interests reported. Supplementary Files ESM1.mpg ESM2.mpg SupplementaryInformarion.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1486763","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":93508244,"identity":"867fd017-1572-4ef9-a225-a5c9e6ab957f","order_by":0,"name":"Elias Silva Santos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYDACdsYGECXHwHyGgQHCJgSYIcqMGdhyiNYCoRIbiNbCz8zc+PHHn7r0Dcd4D35g3HGPsBbJZsZmad62w7kbjvElSzCeKSasxeAwY4M0Y8OB3A33ewwkGNsSCGuxP8zY/BPkMINjPMY/iNJiwMzYJsHDxpwA1GJGnC0ShxnbrIF+MZx5jC/NIvEMEVr429sf3wQ6TJ7vGO/hGx93EKEFFZCsYRSMglEwCkYBdgAA8R84XKBDw9EAAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal da Bahia","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Elias","middleName":"Silva","lastName":"Santos","suffix":""},{"id":93508247,"identity":"75ac0523-fb7d-42ba-8cb3-5b29d7f508c0","order_by":1,"name":"Elias Ramos Souza","email":"","orcid":"","institution":"Instituto Federal de Educação, Ciência e Tecnologia da Bahia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elias","middleName":"Ramos","lastName":"Souza","suffix":""}],"badges":[],"createdAt":"2022-03-24 20:29:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1486763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1486763/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19731006,"identity":"ca4d172f-7099-425b-9e74-cc6442957ffe","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51150,"visible":true,"origin":"","legend":"\u003cp\u003eOverlapping of the FMN in redocking simulation. Docked FMN coloured green. The figure was visualized with VMD\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/34721e8ecba512c41a6bed08.jpg"},{"id":19731007,"identity":"8fcd48c2-fad7-40f4-a60c-7cb576b8938e","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42183,"visible":true,"origin":"","legend":"\u003cp\u003eSubstrates attached to the hydrophobic cavity of \u003cem\u003eladA\u003c/em\u003e a) represents the interaction FMN-linoleic acid and b) represents the interaction FMN-palmitic acid. The figure was visualized with VMD\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/e1a17de36f37d3e6063b86e8.jpg"},{"id":19731010,"identity":"d96737f6-f7fe-4f36-907f-7611507e2203","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":248071,"visible":true,"origin":"","legend":"\u003cp\u003ea) RMSD of systems concerning optimised structure as a function of time simulation. It is observed large fluctuations in the system LAD_F. b) Projection of the motion of the backbone atoms in LAD_P, LAD_L, and LAD_F in phase space along the first two principal eigenvectors. c) The\u0026nbsp;loop mobility of exposed to the solvent and active site regions in LAD_F (green), LAD_P (black), and LAD_L (red)\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/7fc9e1469e36dd866c086d6f.jpg"},{"id":19731013,"identity":"08e54f27-dd9b-45ca-a28c-5ff06abdb550","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31645,"visible":true,"origin":"","legend":"\u003cp\u003eFunction radial distribution of water around the centre of mass of the FMN\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/8c23ba206fae3d849d649dcc.jpg"},{"id":19732184,"identity":"72ea7508-fb2d-4702-944b-481802e74766","added_by":"auto","created_at":"2022-03-29 13:12:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110744,"visible":true,"origin":"","legend":"\u003cp\u003ea) Tunnel inferred from the webserver MoleOnline. b) The profile depicts polarity and charges along the tunnel of the enzyme where the channel is in an open conformation. The figure was visualized with LiteMol\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/3a5b1e325013773eb51d6232.jpg"},{"id":19732186,"identity":"adad843b-3ddf-43f6-a6fd-f7a6c2b88d2c","added_by":"auto","created_at":"2022-03-29 13:12:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84425,"visible":true,"origin":"","legend":"\u003cp\u003eComparative root mean square fluctuation-RMSF of the systems\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/a141d139eb35b843f22d5131.jpg"},{"id":19733054,"identity":"e08955ff-e7d8-4472-9480-d8c6bdadaa37","added_by":"auto","created_at":"2022-03-29 13:17:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":56846,"visible":true,"origin":"","legend":"\u003cp\u003eOxygen atom highlighted in yellow line inserted into the a) palmitic acid and b) linoleic acid. The figure was visualized with VMD\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/6d585cf2c6140f0002772b7d.jpg"},{"id":20740024,"identity":"9890c68a-5931-4e8a-8b98-8528e50ff4e2","added_by":"auto","created_at":"2022-04-25 17:29:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":670218,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/b45ab3c1-7315-4229-ae9f-7122c388cae5.pdf"},{"id":19731014,"identity":"a0f07fa7-b598-4aae-beb3-11715dbb52e9","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"mpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":598972,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.mpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/c61711b63f61d281ed74b712.mpg"},{"id":19732187,"identity":"cd9c8932-45a2-47e2-bb9b-001dbb9aacef","added_by":"auto","created_at":"2022-03-29 13:12:00","extension":"mpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":598265,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.mpg","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/ae9456ed63f3d27c0d05dc5f.mpg"},{"id":19731016,"identity":"ae7dc42d-b313-42a7-839b-194ffc4b381d","added_by":"auto","created_at":"2022-03-29 13:07:00","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":391993,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformarion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1486763/v1/dbe7b67dda8dce0696dd9b22.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alkane monooxygenase study by molecular modelling techniques","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePetroleum dependence as an energy source has become increasingly important, especially in emerging countries, which contribute to the acceleration of global climate change due to excessive emissions of particulate matter and greenhouse gases. This truth has led many nations to seek new alternatives for energy production, wherein biodiesel has emerged as one of the main renewable energy resources in its energetic matrix. According to Gucciardi [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], there is a growing tendency to exchange fossil fuels for biofuels. In pure form or, more commonly, in diverse proportions combined with traditional oil-derived diesel [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A few years ago, Brazil adopted the utilisation of blend biodiesel and diesel for economic and environmental purposes. The whole substitution of diesel by 100% vegetable oil is not applicable, because pure oil as a fuel has two severe problems: one is oil deterioration and the other is incomplete combustion in diesel engines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, factors such as elevated temperature and the presence of light or extraneous materials such as metals or initiators [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] influence the self-oxidation and nature of reactions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A relevant issue in the productive chain is the biodegradation of petrol diesel that has caused serious environmental and economic problems, mainly due to corrosion or blockage of pipelines in the storage. The biodiesel predominantly consists of eight different fatty acids methyl-esterified as oleate, palmitate, linoleate, laureate, and linoleic acid. The methyl esters present in biodiesel have a comparatively high octane number and are considered a biodegradable alternative to recalcitrant gasoline oxygenates [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nevertheless, because fatty acids and aromatic compounds are of high relative molecular weight, some microorganisms have demonstrated the potential to degrade biodiesel more easily than petroleum diesel [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In some cases, metabolic activity can change the chemical sort of the compound (biotransformation) but not lead to complete mineralisation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Biofuel contamination by microorganisms, especially in storage systems, primarily occurs in the presence of water in storage tanks [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The water absorbed by biodiesel through flushing enhances the oxidation and compromises the quality of the fuel stored, clogging of filters and nozzles due to the condensation reactions involving double bonds, which results in the formation of gums and sediments, increasing the viscosity of biodiesel [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Microbes activate alkanes under aerobic or anaerobic conditions with different enzyme systems [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Under aerobic conditions, oxygen serves as the electron acceptor whereas under anaerobic conditions; sulphate and nitrite accept electrons to complete the processes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The whole reaction catalysed by flavoprotein monooxygenase involves three chemical processes: a) NAD(P)H reduction of flavin; b) reaction of the reduced flavin with O\u003csub\u003e2\u003c/sub\u003e to produce a C\u003csub\u003e4a\u003c/sub\u003e-flavin [hydro] peroxide; and c) binding, orienting and activating the substrate for oxygenation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, the mechanism of transferring labile reduced flavin from the reductase to the oxygenase is unknown [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and the reduced enzyme-bound flavin reacts with oxygen to form C\u003csub\u003e4a\u003c/sub\u003e \u0026ndash; (hydro) peroxyflavin, which is stable in the absence of substrates [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The aerobic biodegradation of a hydrocarbon is a process that depends on several factors, from the number of hydrocarbons to the quantities of oxygen molecules present in the environment [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In many ecosystems, there is already an adequate indigenous microbial community capable of extensive oil biodegradation, if environmental conditions are favourable for oil-degrading metabolic activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A group of microorganism enzymes that use carbon as an energy source mediates the nucleophilic attack of the aliphatic chain or aromatic parts of hydrocarbon culminating in the production of primary alcohols [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this approach, alkanes are first attacked at their terminal methyl group to yield the corresponding primary alcohols, which are further oxidised by alcohol and aldehyde dehydrogenases [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The terminal oxidation pathway undergoes ω-hydroxylation at the terminal methyl group (the ω position); yielding ω-hydroxy fatty acid that is further converted to a dicarboxylic acid, which then also enters β-oxidation [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, it has been suggested that an oxygen-independent formation of a terminal double bond occurs as an alternative mechanism during aerobic growth on alkanes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The three-dimensional structures of long-chain alkane hydroxylases remained unclear until the 2.7 \u0026Aring; apoenzyme and 1.9 \u0026Aring; holoenzyme structures of \u003cem\u003eladA\u003c/em\u003e, a long-chain alkane monooxygenase to be reported in 2008 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The \u003cem\u003eladA\u003c/em\u003e was isolated from the thermophilic bacillus \u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e NG80-2, which utilises terminal oxidation as a pathway for the conversion of long-chain \u003cem\u003en\u003c/em\u003e-alkanes. Alkane monooxygenase is one of three enzymes involved in the process of aerobic biodegradation. It is a homodimer composed of (β/α8) barrel fold capable of degrading alkanes for the conversion of long-chain n-alkanes from C\u003csub\u003e15\u003c/sub\u003e to at least C\u003csub\u003e36\u003c/sub\u003e by introducing oxygen into the substrate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The structure of \u003cem\u003eladA\u003c/em\u003e contains a triose phosphate isomerase (TIM) barrel fold that differs from the prototypical TIM barrel structure due to five extended insertion regions and an extension at the C-terminus of the polypeptide chain [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A pocket at the C-terminal entrance of the TIM barrel is sufficiently large to accommodate FMN, O\u003csub\u003e2\u003c/sub\u003e, and part of the terminal of a long-chain \u003cem\u003en\u003c/em\u003e-alkane. The flavin ring of FMN is located in the barrel and its S\u003cem\u003ei\u003c/em\u003e-face is exposed to the solvent [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The ribityl side chain and phosphate moieties are inserted between strands β4 and β5 of \u003cem\u003eladA\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A cavity above the \u003cem\u003eSi\u003c/em\u003e-face of FMN consists of residues Phe10, Met12, His17, Ala57, Val59, Tyr63, Gln79, His138, His311, Trp348, and Phe349 with polar residues concentrated on the left sphere and hydrophobic residues on the right sphere [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. When supplied by different enzyme systems, through the reaction of the reduced flavin with O\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eladA\u003c/em\u003e provides C\u003csub\u003e4a\u003c/sub\u003e-flavin (hydro) peroxide activating the substrate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This enzyme creates a highly potent oxidizing agent from oxygen in the active site, which overcomes the low reactivity of the hydrocarbons and allows the introduction of functional groups that enable further biochemical conversions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlkane hydroxylases are widespread in petroleum-degrading bacteria and a number of them are efficient at oxidising substrates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Hence, there is considerable interest in employing alkane hydroxylases for industrial applications. Thermophilic long-chain \u003cem\u003en\u003c/em\u003e-alkane-degrading bacterial strains are of particular interest for their biotechnological applications [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although many of the \u003cem\u003eGeobacillus\u003c/em\u003e come from high-temperature environments, it has been pointed out that they are frequent in temperate-climate soil environments where they are unable to grow due to temperature restrictions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, there are several advantages to using thermophilic microorganisms in the bioremediation of hydrocarbons over mesophilic organisms. For instance, the production of biosurfactants that act as solubilising agents. As such, \u003cem\u003eladA\u003c/em\u003e is a perfect target for study, especially as an alternative to recovery areas affected by oil spillage. So far, few alkane hydrolases representatives of long-chain have been characterised. Then, this work has the goal of supplying new insight into the dynamics and structural changes of \u003cem\u003eladA\u003c/em\u003e extracted from \u003cem\u003eGeobacillus thermodenitrificans\u003c/em\u003e NG80-2, by introducing two aliphatic compounds of biodiesel as substrates and O\u003csub\u003e2\u003c/sub\u003e molecules for more explanation of the \u003cem\u003eladA\u003c/em\u003e structure and the effects of dissolved oxygen in a salt environment. Here, a hybrid quantum mechanics/molecular mechanics (QM/MM) approach simulation is applied for the investigation of the catalytic mechanism of oxidation involving FMN and substrate interaction. A computational estimate of the binding free energy was also presented to elucidate the interaction formed between the cofactor FMN and the substrate.\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Molecular docking\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.1.1 Preparation of the receptor and ligands\u003c/h2\u003e\n \u003cp\u003eThe structure of \u003cem\u003eladA\u003c/em\u003e obtained from the Protein Data Bank (PDB CODE 3B9O) is a target for study. Autodock Tools [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] was used to create a model of the A chain by introducing hydrogen and its protonation states were optimised. Three structures of linoleic acid, COD-CID 5280450, palmitic acid COD-CID 985, and FMN COD-CID 643976 were obtained from the PubChem database (pubchem.ncbi.nlm.nih.gov/) in the SDF file format. We used OpenBabel software version 2.3.1 [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] to convert them into PDB format. All bonds were defined to be rotational and bonded using the Autodock Tools for protonation states and BCC method implemented on ACPYPE [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] calculated partial charges. The binding site selected for docking is formed by including a group of residues based on knowledge of the amino acids that constitute the active site, such as His17, Met12, Ala57, Val59, Tyr63, Gln79, His311, His138, and FMN. We deleted all structural waters except Wat528 and Wat575 because the presence of these water molecules plays an important role in the accuracy of ligand-protein docking predictions in this system.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.2 Redocking\u003c/h2\u003e\n \u003cp\u003eRedocking simulation was performed by removing the FMN from the \u003cem\u003eladA\u003c/em\u003e structure and reconnecting it to the protein to adjust the docking parameters using AutoDock Vina [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The box simulation was defined around the coordinates of the FMN centre (-26.391, 35.045, 19.412) with a size of 25 x 25 x 25 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e and a grid spacing of 0.375 \u0026Aring; was established. Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e summarises the best parameters achieved with such a procedure. The \u0026lsquo;energy range\u0026rsquo; describes the maximum energy difference between the best binding mode and the worst one displayed (given in kcal/mol); \u0026lsquo;num modes\u0026rsquo; parameter corresponds to the maximum number of generated binding modes. The \u0026lsquo;exhaustiveness\u0026rsquo; associated with the thoroughness of the search is roughly proportional to the time and \u0026lsquo;seed\u0026rsquo; of the random number generator. The most accurate result was compared with the FMN crystallography position as deduced from the superposition of the two structures (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The evaluated RMSD value (2.04\u0026Aring;) ensures that molecular docking represents a good conformation.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParameters used to set docking\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 70.8333%;\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 29.1667%;\"\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 70.8333%;\"\u003e\n \u003cp\u003eEnergy range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 29.1667%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 70.8333%;\"\u003e\n \u003cp\u003eNum modes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 29.1667%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 70.8333%;\"\u003e\n \u003cp\u003eExhaustiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 29.1667%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 70.8333%;\"\u003e\n \u003cp\u003eSeed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 29.1667%;\"\u003e\n \u003cp\u003e-5594\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.3 Protein-ligand docking\u003c/h2\u003e\n \u003cp\u003eAutoDock Vina accomplished the docking of linoleic acid and palmitic acid into \u003cem\u003eladA\u003c/em\u003e. The best binding mode procedure obeyed two steps: identical solutions in terms of conformation were separated and results with lower energy allocated in families. The selection of complex used as a template for molecular dynamics was based on the hydrogen bond number between FMN and substrate among those with lower binding energy. The complexes were visualised using VMD 1.9.1 [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] and submitted to optimisation using the Steepest Descent algorithm implemented in GROMACS 5.1.2 [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.4 Classical molecular simulation\u003c/h2\u003e\n \u003cp\u003eFor the models obtained from the docking procedure, classical molecular dynamics simulation was performed using the Charmm27 force field [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] implemented in the GROMACS package. The topologies of linoleic acid, palmitic acid, and FMN were obtained from the server Web SwissParam [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] and partial charges were assigned through ACPYPE. The LINCS algorithm [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] applied to all bonds in the complex allowed 1 fs time steps and the SETTLE algorithm [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] for water molecule bonds. The cut-off of 1.1 nm was used to calculate non-bonded interactions, resorting to the PME method [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. The V-rescale method [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] coupled with a time constant of 1 ps kept the temperature at 300 K, while the Parrinello-Rahman method [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e] with 5 ps time constant relaxation to remain the pressure of 1 bar. For this study three systems were built: The primary system, named LAD_L (with linoleic acid) was soaked in a box of volume 4356.49 nm\u003csup\u003e3\u003c/sup\u003e with ~\u0026thinsp;17000 water molecules of TIP3P type and 34 oxygen molecules distributed randomly around the protein surface. We made the O\u003csub\u003e2\u003c/sub\u003e concentration low to make sure it does unsaturate in water at ambient conditions, warranting enough samples of O\u003csub\u003e2\u003c/sub\u003e to the pathway in the protein. The margin of 1.2 nm between protein and every side of the box as well as for the second system labelled LAD_P (with palmitic acid). The third system, called LAD_F was made equivalently, but without substrate or oxygen molecules. NaCl molecules at 0.15 M concentration neutralise the systems by equilibrating them during 40,000 ps with a restriction position for protein. Then, we ran simulations for each system for 200,000 ps, while the Maxwell\u0026ndash;Boltzmann distribution generated initial velocity and Xmgrace version 5.1.22 built graphs. For obtaining an insight into the binding energy between FMN and the substrate, we performed a calculation based on Poisson-Boltzmann surface area (MM-PBSA) [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. This method combines three energy terms to explain the change in the binding free energy according to the equation:\u003c/p\u003e\n \u003cp\u003e\u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e = \u0026lang;G\u003csub\u003ePL\u003c/sub\u003e\u0026rang; - \u0026lang;G\u003csub\u003eP\u003c/sub\u003e\u0026rang; - \u0026lang;G\u003csub\u003eL\u003c/sub\u003e\u0026rang;, (1)\u003c/p\u003e\n \u003cp\u003eG\u003csub\u003ePL\u003c/sub\u003e, G\u003csub\u003eP\u003c/sub\u003e and G\u003csub\u003eL\u003c/sub\u003e are the total free energy of the protein-ligand complex, the total free energies of the isolated protein, and the ligand in a solvent, respectively. Eq.\u0026nbsp;(1) is estimated from\u003c/p\u003e\n \u003cp\u003e\u0026Delta;G\u003csub\u003ebind\u003c/sub\u003e=E\u003csub\u003ebond\u003c/sub\u003e + E\u003csub\u003eele\u003c/sub\u003e + E\u003csub\u003evdw\u003c/sub\u003e + G\u003csub\u003epol\u003c/sub\u003e + G\u003csub\u003enp\u003c/sub\u003e -T\u0026Delta;S, (2)\u003c/p\u003e\n \u003cp\u003eThe first three terms are the MM energy terms from bonded i.e., bond, angle, and dihedral. The third and fourth terms refer to electrostatic and van der Waals interactions. G\u003csub\u003epol\u003c/sub\u003e and G\u003csub\u003enp\u003c/sub\u003e are the polar and non-polar contributions to the solvation free energies, and T\u0026Delta;S is the entropy term. For configurational entropy calculation, we used the Schlitter method [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.5 Quantum mechanics (Reaction path calculation)\u003c/h2\u003e\n \u003cp\u003eTwo steps were considered in the oxidation reaction: the first step is the reaction of O\u003csub\u003e2\u003c/sub\u003e with reduced FMN, and the second one is the reaction of the O atom with the substrate. We performed the activation energy calculations with the nudged elastic band (NEB) method [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], implemented in Quantum Espresso suite version 6.0 (QE) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. NEB creates a set of images of the system connected with springs to make a representation of a configuration path. In estimating activation energy, the representative initial structure selected at the latter time of the trajectory was obtained from GROMACS. Due to computational limitations, executed calculations in the subsystem containing the substrate, FMN and O\u003csub\u003e2\u003c/sub\u003e because of the expensive computations. Reactant geometries were estimated by optimising the geometry with the conjugate gradient algorithm implemented on QE. The climbing image method was used for the highest energy image, increasing the accuracy of the calculations [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e] and fixed all atom positions in the system except for the atoms of the O\u003csub\u003e2\u003c/sub\u003e molecule. In addition, we considered for both systems the following reaction coordinates r\u0026thinsp;=\u0026thinsp;d (C\u003csub\u003e4a\u003c/sub\u003e-O - CH/substrate) as shown in the Table (Online Resource 1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e3.1 Docking simulations\u003c/h2\u003e\n \u003cp\u003eThe characterisation of \u003cem\u003eladA\u003c/em\u003e and substrates through docking simulation shows the ways of bonding the substrates attached to the cavity of the hydrophobic \u003cem\u003eladA\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The results were analysed based on the lowest binding energy values. The findings have shown that hydrogen bond plays a critical role in the stabilisation of the substrates in both complexes, but some hydrophobic contacts and salt bridges help to equilibrate them into the active site. In addition, the water molecules (Wat528 and Wat575) are involved in protein-substrate recognition mediating hydrogen bonds between the protein and the substrate. Linoleic acid is perfectly bound within the active region by hydrogen bonds, which produces energy of -0.73 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-3.05 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Added to that, hydrophobic contacts with Ile18, Thr316, Trp348, Phe349, and salt bridge help counteraction it. Likewise, palmitic acid maintained steady to the active site thanks to the energy of -0.65 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-2.72 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) provided by hydrogen bonds. Hydrophobic contacts with Trp348, Phe349, and bridge salt collaborate with the balance. Nevertheless, the data show that the total electrostatic interaction has a smaller influence on the stabilisation of substrates, i.e. -0.035 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-0.14 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for the linoleic acid and 0.043 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.18 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) palmitic acid. The H\u003csub\u003e5\u003c/sub\u003e atom bonded to the N\u003csub\u003e5\u003c/sub\u003e atom of the isoalloxazine ring directly points to the H atom of the carboxyl group of linoleic acid, the binding energy of -5.52 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-23.10 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (average distance 2.3 nm) and \u0026minus;\u0026thinsp;5.41 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-22.64 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for palmitic acid whose average distance is 2.7 nm. Van der Waals interaction \u0026minus;\u0026thinsp;8.54 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-35.73 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (linoleic acid) and \u0026minus;\u0026thinsp;7.81 kcal.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-32.68 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (palmitic acid) is responsible for maintaining the two substrates firm in the complexes. In summary, the docking results show the significance of van der Waals interaction for the stabilisation of the substrates within the active region of \u003cem\u003eladA\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e3.2 Molecular dynamics\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.2.1 Overall structure\u003c/h2\u003e\n \u003cp\u003eAfter identifying the best complex from molecular docking, molecular dynamics simulation was used to estimate the root-mean-square deviation (RMSD) of the protein for the quantification of the degree of conformational changes during MD simulations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The analysis of protein stability was fulfilled inspecting the conformational states takes into account the changes in the position of atoms concerning the optimised structure, which revealed that LAD_L and LAD_P have relative stability around 0.22 nm and 0.30 nm, respectively, as can be seen through some plateaus in their trajectories. Alternatively, the LAD_F plot presented the highest increase in RMSD during the last 100,000 ps of the simulation, which shows many conformational changes as a reflection of local fluctuations in some properties of the protein, such as shape and size. Therefore, the stability of \u003cem\u003eladA\u003c/em\u003e is altered when substrates bind, inducing changes in structure and conformational flexibility. We also performed the principal component analysis (PCA), where the set eigenvectors were obtained from backbone trajectories during the same period of simulation used to calculate the RMSD through the projection of two principal components (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The data show higher dispersing in LAD_P confirming the great flexibility of the loop regions exposed to the solvent, wherever one of these loops works as a lid for a tunnel of access to the active site as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec. Additionally, the loop in the active site region constituted by residues Ala57, Asp58, Val59, Val60, and Gly61 undergoes different conformational arrangements induced by substrates. For example, the stretched conformation and orientation in the last picoseconds of simulation in LAD_P increases the contact with Asp348. In contrast, the orientation of the linoleic acid reduces its contact with Asp348. As such, the substrate depends on the conformational changes in the loop to be catalysed. The RDF-radial distribution function represented by g(r) in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e displays the distribution of water around the centre of mass of the FMN. The first hydration shell consists of water at 0.18 nm and the second one occurs at 0.21 nm. The movement of water to greater distances from the FMN caused by substrates gradually assumes the bulk of the water from this region. Hydrogen bonds formed with amino acids from the active site plus water molecules around the isoalloxazine ring and ribityl group, water bridges, and hydrophobic contacts maintain FMN tight in the packed hydrophobic core. This result suggests the formation of a hydrophobic environment surrounding FMN and the three water found there should play an important role in the catalytic process. As shown in the Figure (Online Resource 2), the number of these contributions is a limiting factor for conformational changes or loss of vibrational entropy thanks to the constraints of binding.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.2.2 Diffusion of O\u003csub\u003e2\u003c/sub\u003e molecule into \u003cem\u003eladA\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eDiffusion and reaction conversion of O\u003csub\u003e2\u003c/sub\u003e requires the initial concentration for substrate biodegradation. Many enzymes, such as Cytochrome cOxydases [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e] use molecular oxygen to explore tunnels for controlling specific substrates or transport ions from the surface of the protein to the active site, acting as pathways for substances between the bulk solvent and the active site. The \u003cem\u003eladA\u003c/em\u003e has many tunnels to transport oxygen molecules, considering that oxygen molecule is transient at the active site, but few are used effectively. This section presents results obtained from the analysis of possible tunnels for oxygen molecule transport through \u003cem\u003eladA\u003c/em\u003e, utilising the MOLE Online web server [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. The O\u003csub\u003e2\u003c/sub\u003e explores some cracks on the surface of the protein to find cavities formed mainly by hydrophobic residues. The results insinuate the existence of many tunnels, but four are probable for transport because of their low curvature and short length (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). In analysing these tunnels, the T1C1 tunnel connected to the T2C1 is lined by Tyr66, Arg67, Ser69, Asp71, Thr72, Arg75, Ala310, Tyr63, Val65, and His318, Gly314, Gly317, respectively. However, they are directed away from the active site. T3C1 tunnel covered by residues His311, Tyr312, Thr316, Trp348, Phe349, Gly347, and Gly315 is wide enough to allow the substrate to reach the catalytic centre. Nevertheless, as oxygen is a hydrophobic molecule, it must prefer hydrophobic channels where it can use the dynamics structure to reach the active site spreading through T4C1 coated by residues Val59, Val60, Asp58, Asp84, Phe105, Ser137, Ser106 and length of 16.5\u0026Aring; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Therefore, these data reveal that oxygen molecules and the substrate use distinct routes for reaching the reactive centre. For alternative tunnels, they seem unlikely because longer side-chains can obstruct the passage of O\u003csub\u003e2\u003c/sub\u003e due to conformational changes or are not hydrophobic routes. In Lad_L, the first oxygen enters the active site at 6,311 ps and remains there for a long time. However, in Lad_P the first oxygen reaches the active site cavity at half time of simulation, although many more oxygen molecules come to the active site in both systems. Translational diffusion of oxygen molecule into the active site was computed and shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e at the interval of 100,000\u0026ndash;150,000 ps. According to data, the largest flux of oxygen molecules is found to be in the x-direction (LAD_P) and y-direction (LAD_L). This behaviour seems to be due to the substrate length and conformational changes of the residues around the substrate. The diffusion decreases with obstructions caused by solidary movement of some residues from the tunnel or by water bridge formation as a response to substrate conformational changes. Hence, the amino acids that compose the tunnel play an important role in the diffusivity of the oxygen molecule and, consequently, the capacity of the enzyme to degrade substrates. According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the fluctuations of the protein residues (RMSF) in the systems were virtually similar and certain residues of the active site have highly fluctuated. Comparing the RMSFs for the binding site of systems, we can see that, on average, the amino acids bind the ligand and keep them firm throughout the simulation. In LAD_P, Tyr63 shows more fluctuation than LAD_L or LAP_F, but surprisingly, His311 and His138 fluctuated less in the bounded systems than in the unbounded. Additionally, when the histidines in bounded systems are compared, the fluctuation is major in LAD_P, suggesting that the binding of a certain substrate cannot stabilise the complex. Thereby, speculation by Liu et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] that the substrate-binding mode would require substrate specificity and determine the range of alkane chain length for catalysis finds support. Carbon-chain lengths of C\u003csub\u003e14\u003c/sub\u003e and lower would be so short that the terminal carbon could not reach the active site while the opposing terminal part was anchored to the protein [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast, carbon-chain lengths of C\u003csub\u003e15\u003c/sub\u003e and higher would bind with the exact carbon chain fitting from the active site to the anchoring site. Indeed, the findings of Owsianiak et al. [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e] using a consortium of bacteria isolated from an oil-contaminated site showed complete degradation of methyl-esters in blends of biodiesel fuels, and more than 50% C\u003csub\u003e16\u003c/sub\u003e (palmitic acid) was degraded. In the same way, Miller and Mudge [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e] observed that unsaturated (C\u003csub\u003e18\u003c/sub\u003e) fatty acids were degraded more rapidly than the methyl-esters of saturated fatty acids. Therefore, biodegradability has to be a function of chain length, conformation, degree of substitution (unsaturation), C\u0026thinsp;=\u0026thinsp;C position, and cis and trans configurations [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe oxygen diffusion coefficient in different directions\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(x10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003ecm\u003csup\u003e2\u003c/sup\u003e.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAD_P\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAD_L\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003ex\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003ey\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003ez\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e*The standard error of 0 is not shown\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.2.3 FMN binding\u003c/h2\u003e\n \u003cp\u003eChanges in the angles formed between the atoms (C\u003csub\u003e4a\u003c/sub\u003e-N\u003csub\u003e10\u003c/sub\u003e-C\u003csub\u003e10\u003c/sub\u003e) named as plane 1 and (C\u003csub\u003e9A\u003c/sub\u003e-C\u003csub\u003e5A\u003c/sub\u003e-C\u003csub\u003e10\u003c/sub\u003e) as plane 2 and the distance between (N\u003csub\u003e10\u003c/sub\u003e-C\u003csub\u003e4\u0026apos;\u003c/sub\u003e) atoms of the isoalloxazine ring were monitored and summarised in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. In terms of classical molecular dynamics, the bending motion of the isoalloxazine ring is practically the same for all systems. Some residues Gly315, His154, Asp155, and Ser230 interact with FMN tail making hydrogen bonds that restrict the movement of tail keeping the average distances between N\u003csub\u003e10\u003c/sub\u003e-C\u003csub\u003e4\u0026apos;\u003c/sub\u003e around 0.18nm (Lad_P and Lad_F) and 0.23 and Lad_L. However, as shown below, an examination of the \u003cem\u003eab initio\u003c/em\u003e results in both bonded systems revealed that the structure of the isoalloxazine ring displayed some distortion to your original plan, which is not revealed by classical simulations because the force field is not capable to show this distortion, caused by the electronic change in the isoalloxazine ring. The results indicate plane 1 rotates to 121.91\u003csup\u003e◦\u003c/sup\u003e (LAD_P) and 90.75\u003csup\u003e◦\u003c/sup\u003e (LAD_L) while plane 2, 93.80\u003csup\u003e◦\u003c/sup\u003e (LAD_P), and 77.95\u003csup\u003e◦\u003c/sup\u003e (LAD_L) in favour of substrate cleavage. As such, changes in the isoalloxazine ring modulate the electron transfer process producing different orientations and distances between the reactive centre and the substrate, improving the electrostatic interaction between them. According to the proposed mechanism by Liu et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], His138 must act as a proton donor in the reaction between reduced flavin and an oxygen molecule that leads to the formation of the C\u003csub\u003e4a\u003c/sub\u003e-hydroperoxyflavin intermediate. Direct proton transfer requires the proximity of the proton donor and acceptor to be within the distance of a typical H-bond (\u0026sim;0.28 nm between heavy atoms) [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here, and for geometric reasons, His138 cannot directly transfer a proton to the isoalloxazine ring (0.35\u0026ndash;0.69 nm distance) unless the presence of water molecules near the isoalloxazine ring is imperative. According to the data obtained in these simulations, His138 helps to steady the ribityl group and the phosphate group with one water bridge in LAD_P (please, see again Online Resource 2). Additionally, the same data suggest that transfer could occur among isoalloxazine ring, one water and Thr104 in LAD_L or by a hydrogen bond formed with Asp58 in LAD_P owing to specific changes in the protein structure produced by water reorientation or polar side chain positions in the active site [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. That reorientation must change the protonation state during the reaction. In bounded systems, the substrate has some conformational mobility during the simulation coming to the isoalloxazine ring. This approximation displaces the water close to the isoalloxazine ring, creating a hydrophobic microenvironment between them. Linoleic acid is stabilised by Asp58 and Lys347, which can be involved in substrate activation during hydroxylation, and Trp348 steady palmitic acid.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFMN average angles and distances isoalloxazine ring\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAngle (deg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAD_P\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAD_L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAD_F\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlane 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.32 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.38 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.27 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlane 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89.11 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89.17 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.99 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDistance (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.18 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.23 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.18 (\u0026plusmn;\u0026thinsp;0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e*Standard errors are given in parentheses\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2.4 FMN interaction with the substrate\u003c/h2\u003e\n \u003cp\u003eIn this section, the binding free energy change is computed through the MM-PBSA approach for FMN-palmitic acid and FMN-linoleic acid, taking into consideration configurational changes. Although methods to estimate the binding free energy, such as Free Energy Perturbation (FEP) or Thermodynamic Integration (TI) have more accuracy they are computationally expensive, thus we used MM-PBSA approach for calculation. The binding free energy was estimated by calculating the configurational entropy of the systems, where the dielectric constant (ɛ = 4) was assumed as the default to represent the internal dielectric constant. GROMACS tool computed the external dielectric constant whose value found was ɛ =101.07 and Schlitter method computed the configurational entropy. The configurational entropy found was 7534.66 J. (mol. K)\u003csup\u003e\u0026minus;1\u003c/sup\u003e in Lad_L and 1363.93 J. (mol. K)\u003csup\u003e\u0026minus;1\u003c/sup\u003e in Lad_P, where the loss entropic effect is smaller in Lad_P than Lad_L. The binding free energies between FMN-Palmitic acid and FMN-linoleic acid were estimated at -15.77 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u0026minus;\u0026thinsp;12.13 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. These values are different from those obtained by docking refining the results of the scoring function. Van der Waals energies of -45.53 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (LAD_P) and \u0026minus;\u0026thinsp;64.09 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (LAD_L) were critical contributions for binding free energies. The MM-PBSA approach must provide results of intermediate quality comparatively to the methods aforementioned (FEP and TI) for calculating binding free energy change, but it gives an idea of the binding energy between FMN and the substrates here studied, improving the results obtained through molecular docking simulations.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.3 Studies of quantum mechanics\u003c/h2\u003e\n \u003cp\u003eIn this moment of the study, an approach using quantum mechanics calculation was employed to investigate the oxidation mechanism for substrate mediated by oxidation of the FMN.\u003c/p\u003e\n \u003cp\u003eKinetic and chemical analysis suggest an initial electron transfer from singlet reduced flavin towards triplet O\u003csub\u003e2\u003c/sub\u003e to make a caged radical pair [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. After spin inversion, the intermediate generated can collapse into a peroxyflavin that is unstable in water and heterolytically dissociates into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and oxidised flavin [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Based on that reaction scheme, we took the last frame of GROMACS simulation to compute path reaction. The results show the first step of the bond formed between O\u003csub\u003e2\u003c/sub\u003e and C\u003csub\u003e4a\u003c/sub\u003e of the isoalloxazine ring has an activation energy of 0.76 eV (74.29 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), i.e., the amount of energy required the reaction to occur. This energy reveals a high cost for translationing the O\u003csub\u003e2\u003c/sub\u003e towards the C\u003csub\u003e4a\u003c/sub\u003e atom of the isoalloxazine ring, where two local minima and a saddle can be seen in the graph in the Online Resource 3. In addition, an experiment performed by Hasting and Gibson indicates that a reaction involving the oxidation of reduced flavin yields activation energy in appearance luminescence is about 0.56 eV (54.4 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIn the second step of the process, the oxygen atom activates palmitic acid with an energy of 0.61 eV (59.82 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and linoleic acid 0.66 eV (64.64 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The movement of the oxygen atom towards the substrate is shown in the animations ESM_1 and ESM_2 in the Online Resource, respectively, which help to clarify the pathway of the reaction. These findings were compared with the findings of Adachi et al., who estimated the activation energy of 50.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in a study with methylated linoleic acid in Autoxidation of Polyunsaturated Fatty acids [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Ishido et al. investigating the oxidation of linoleic acid, in the presence of saturated acids or its methyl esters, found an activation energy of 54.4 kJ.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. In another investigation conducted by Fu et al., the palmitic acid-deoxygenating at a high temperature found 79\u0026thinsp;\u0026plusmn;\u0026thinsp;5 kJ.mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of activation energy [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. Furthermore, Hermida et al. reported an activation energy of 111.57 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for palmitic acid when using nickel as a catalyst for palmitic acid deoxygenation to synthesise n-pentadecane and 1-pentadecene [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. Altogether, we consider that data obtained here are in relative concordance with experimental data, even not taking into account all the complexity of the active site. Although the activation energies calculations using SCF algorithms are well-known to have difficulties when attempting to enforce specific electron distributions [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e], and therefore, encountering convergence difficulties may not point to an intrinsic instability of those pairs, but simply be a consequence of numerical/algorithmic difficulties [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eEnergetic minimisation performed in both systems respecting the reactant state using \u003cem\u003eab initio\u003c/em\u003e dynamics relaxed the bonds ( Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) with the coupling of the oxygen atom between carbons in the acids allowing movement of atoms of acids. In LAD_P, the distance increased from 0.18 nm to 0.27 nm, while in LAD_L increased from 0.17 nm to 0.25 nm.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn this research, it has been possible to elucidate some dynamic and structural aspects of \u003cem\u003eladA\u003c/em\u003e. We investigated some characteristics of the mechanism of \u003cem\u003eladA\u003c/em\u003e degradation through the calculation of QM/MM. The results indicate that water provides a trail for charge transfer for FMN via hydrogen bonds. The reaction paths of oxygen molecules produce new information about the oxidation of FMN and substrates, where the data is close to the experimental findings, although it was not considered certain amino acids or water in the calculation of activation energies. New conformations of these residues or the presence of water molecules in the calculation must yield energy values that would explain the divergence with the experimental results, besides the limitations of the algorithms used. Indeed, the findings show that the enzyme controls the entrance of oxygen or ligands through conformational changes of the residues that form access tunnels from the enzyme surface, providing a favourable environment for the degradation of the selected substrates. This specific result also suggests that the availability of oxygen molecules in the environment determines the rate of degradation of fatty acid molecules that must be similar to those obtained experimentally.\u003c/p\u003e \u003cp\u003eTherefore, this investigation provides new insight into the enzyme \u003cem\u003eladA\u003c/em\u003e as a good source for developing novel ways to improve production techniques and use for biodiesel that is still increasing in Brazil, representing in 2019 about 9.6% by the energy of diesel consumption, according to IEA World Energy Balance [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. On the other hand, the use of this enzyme can mitigate the effect of pollution and recovery production areas degraded, including a reduction in the soil of toxic residue and improvement in production systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other supports were received during the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElias Silva dos Santos contributed to the conceptualization, formal analysis, methodology, investigation, writing-review, editing and visualization\u003c/p\u003e\n\u003cp\u003eElias Ramos de Souza contributed to the review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarcez Gucciardi CA, de Souza Vianna JN (2009) Brazilian Biodiesel Policy: Social and environmental considerations of sustainability. 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PeerJ. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7717/peerj.2805\u003c/span\u003e\u003cspan address=\"10.7717/peerj.2805\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIEA Bioenergy (2021) Implementation of Bioenergy in Brazil. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.iea.org/policies?country=Brazil\u003c/span\u003e\u003cspan address=\"https://www.iea.org/policies?country=Brazil\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 18 February 2022\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biodiesel, petrol diesel, biodegradation, alkane monooxygenase, simulation","lastPublishedDoi":"10.21203/rs.3.rs-1486763/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1486763/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiodiesel biodegradation has been a limiting factor for its use as an alternative for petrol diesel. Thanks to low structural complexity, a group of enzymes such as alkane monooxygenase (\u003cem\u003eladA\u003c/em\u003e) can readily degrade several fatty acids, but the understanding of the mechanism of degradation is unclear yet. Then, we combined molecular docking and hybrid molecular dynamics (QM/MM) approaches for the study of \u003cem\u003eladA\u003c/em\u003e. The results showed that the bending of the isoalloxazine ring to the original plan enhanced its electrical coupling with a substrate. The flavin mononucleotide-FMN oxidation yields an activation energy of 74.29 kJ.mol\u003csup\u003e-1\u003c/sup\u003e, which is necessary to make an oxygen molecule reactive. In addition, the initial activation energy determined for palmitic acid was 59.82 kJ.mol\u003csup\u003e-1\u003c/sup\u003e and 64.64 kJ.mol\u003csup\u003e-1 \u003c/sup\u003efor linoleic acid. The diffusion of oxygen molecules into the active site is controlled by the movement of some residues that compose the tunnel access or by a water bridge.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Alkane monooxygenase study by molecular modelling techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-29 13:06:57","doi":"10.21203/rs.3.rs-1486763/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef1a8c52-9d06-4f1d-8642-f9685458815b","owner":[],"postedDate":"March 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-25T17:29:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-29 13:06:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1486763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1486763","identity":"rs-1486763","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}