Catalysis in Motion: Large-Scale Domain Alternation Enables Co(III)-C Bond Homolysis in Lysine 5,6-Aminomutase

Catalysis in Motion: Large-Scale Domain Alternation Enables Co(III)-C Bond Homolysis in Lysine 5,6-Aminomutase | 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 Article Catalysis in Motion: Large-Scale Domain Alternation Enables Co(III)-C Bond Homolysis in Lysine 5,6-Aminomutase Shiliang Tian, Khai Pham, Peter Voss, Andrew Poore, Mingxin Liu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5313240/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 Lysine 5,6-aminomutase (LAM) is a radical-based enzyme that catalyzes the reversible migration of an amino group between the C5 and C6 positions of lysine. This reaction is mediated by the coenzymes adenosylcobalamin (AdoCbl) and pyridoxal 5’-phosphate (PLP). Our study investigated the activation mechanism of AdoCbl in LAM. Using cryo-electron microscopy (cryo-EM), we resolved structures of LAM in both its ‘Open’ and ‘Closed’ states, revealing a substantial 64.1-degree rotation of the Rossmann domain upon substrate binding, which shifts the AdoCbl cofactor 25.5 Å towards PLP. This large conformational shift enables Co(III)-C homolysis and adenosine radical (Ado•) formation. Following domain alternation, adenosine is stabilized by a key hydrogen bond network composed of Lys371α, Asp299α, and Glu412α, while PLP is anchored by hydrogen bonds and electrostatic interactions with two Tyr and two Arg residues, securing the substrate lysine in place. The enzyme pocket is precisely designed to ensure the proper orientation and positioning of Ado• and PLP-substrate complex, facilitating efficient hydrogen abstraction from the substrate after the formation of the highly reactive Ado• radical, which is crucial for maintaining catalytic efficiency. This study provided the first structural information supporting the long-existing hypothesis of Rossmann domain rotation as part of the LAM activation process, shedding light on the enzymes' conformational dynamics and illustrating how nature employs domain movements to activate metallocofactors and control radical chemistry. Biological sciences/Biochemistry/Bioinorganic chemistry/Metalloproteins Biological sciences/Biochemistry/Enzyme mechanisms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coenzyme B 12 , or 5’-deoxyadenosylcobalamin (AdoCbl), is a biologically active form of vitamin B 12 and one of the most complex metalloprotein cofactors. 1,2 The organometallic complex features cobalt coordinated by a corrin ring, which is functionalized with several substituents, including a dimethylbenzimidazole (DMB) tail that serves as an axial ligand to the cobalt center. In AdoCbl-dependent enzymes, the axial DMB is often replaced by a histidine residue, which not only coordinates the cobalt but also anchors the cofactor to the protein. Members of the AdoCbl enzyme family share common structural and functional features, including a triose-phosphate isomerase (TIM) barrel domain for substrate binding and a Rossmann domain for AdoCbl binding. 3 AdoCbl-dependent enzymes play crucial roles in several fermentation pathways, particularly in anaerobic organisms. 2 A notable example is the Wood-Ljungdahl pathway, where AdoCbl-dependent enzymes facilitate the conversion of CO 2 into acetyl-CoA, a key coenzyme involved in many metabolic processes. This pathway holds significant potential for biofuel production and carbon sequestration. 4 Additionally, AdoCbl-dependent enzymes have important implications in medical fields. Alterations that impair these enzymes' functionality can lead to severe genetic disorders. For instance, a mutation in the gene encoding methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, a condition characterized by dangerous acid buildup in the body. 5 Furthermore, AdoCbl-dependent enzymes are potential targets for cancer therapies. Cobalamin is an essential co-factor for methionine synthase, a key enzyme in DNA synthesis and repair. Disrupting this process could potentially inhibit cancer cell proliferation. 6 AdoCbl-dependent enzymes catalyze radical-mediated reactions that initiate with the homolytic cleavage of the Co(III)−C bond in the AdoCbl cofactor, generating an Ado • radical, which then abstracts a hydrogen atom from the substrate. 1,3,7 Outside the protein environment, the Co(III)−C bond has a dissociation energy of approximately 30 kcal/mol, corresponding to an estimated bond dissociation rate constant of ~3.8 x 10⁻⁹ s⁻¹. 1 However, the catalytic rate constants of AdoCbl-dependent enzymes are typically in the range from 10 to 100 s⁻¹, which implies a reduced Co(III)-C bond dissociation energy around 13 kcal/mol (Fig. 1). To achieve this remarkable rate enhancement over 10 9 fold, the enzymes must destabilize the Co(III)-C bond by about 17 kcal/mol. Various mechanisms have been proposed to explain the activation of the Co(III)−C bond in the enzymatic environment. Early studies indicated that a trans effect of the histidine, as an axial ligand to the cobalt center, could weaken the Co-C bond. 8-10 However, resonance Raman (rR) measurements showed only a slight shift in the Co(III)-C bond vibration in MCM, from 424 cm⁻¹ in the enzyme-bound form to 420 cm⁻¹ in the free cofactor form, indicating that histidine replacement of the DMB ligand and protein binding only weakens the Co(III)-C bond by about 0.5 kcal/mol. 11 Distortion of the corrin ring has been reported to destabilize the Co-C bond, but primarily in model coplexes. 11-13 In MCM, crystallography studies revealed that in the absence of substrate, the TIM-barrel domain remains open. Upon substrate binding, the barrel closes, displacing the adenosyl group of AdoCbl and facilitating the formation of the Ado • radical. These conformational changes have been calculated to lower the barrier for Co(III)-C bond homolysis by approximately 7 kcal/mol. 14,15 Additionally, electrostatic interactions between the ribose of the adenosyl group and amino acid residues in the MCM's active site are critical for catalysis. Removal of the 2’-OH from the natural cofactor results in a rate decrease of up to two orders of magnitude. 15-17 A tyrosine (Tyr) residue near the AdoCbl cofactor in MCM has been calculated to lower the reaction barrier by 1 kcal/mol via a hydrogen bond network formed between its −OH group, the carboxylate group of the substrate, and a side chain of AdoCbl. 18 This interaction promotes proton-coupled electron transfer (PCET) between the Tyr and AdoCbl, generating a [AdoCbl] •− /Tyr • diradical state. 19,20 While these studies have provided valuable insights into the Co(III)-C homolysis in MCM, the mechanisms driving the 10⁹-fold rate acceleration of Co(III)−C bond homolysis in other AdoCbl-dependent enzymes remain largely unexplained. Beyond Co(III)-C activation, AdoCbl-dependent catalysis also features highly controlled radical chemistry. After the cleavage of the Co(III)-C bond in AdoCbl, the Ado • radical must be positioned to abstract a hydrogen atom from the substrate. This highly reactive, carbon-centered radical is a key intermediate in AdoCbl-dependent enzymatic reactions. In principle, this radical could react with C-H bonds or other functional groups within the substrate-binding site, potentially leading to side reactions that could compromise the integrity of the catalytic site. However, no such side reactions have been observed in AdoCbl-dependent enzymes. How do these enzymes protect the highly reactive Ado • radical during catalysis? How do they ensure reaction selectivity to guarantee the formation of the intended products? What precise structural changes occur during these enzymatic processes? Answering these questions will not only deepen our understanding of the structure-function relationships and mechanisms of AdoCbl-dependent mutases but also enhance their potential as therapeutic targets for various cancers. 6 Lysine 5,6-aminomutase (LAM) is a member of a subfamily of AdoCbl-dependent enzymes that utilizes an additional cofactor, pyridoxal 5′-phosphate (PLP), for catalysis. LAM catalyzes the reversible shift of an amino group between the C5 and C6 positions of D-lysine and L-β-lysine. 21 The x-ray crystal structure of LAM from Clostridium sticklandii ( Cs LAM) reveals that it functions as an α 2 β 2 tetramer. 22 The enzyme employs TIM barrel architectures for PLP and substrate binding, while AdoCbl is coordinated through Rossmann domains. Although PLP resides within the TIM barrel, lysine 144, which is covalently linked to PLP, is from the Rossmann domain. Upon substrate binding to PLP and Co(III)-C bond homolysis, hydrogen atom abstraction (HAA) occurs between the Ado • radical and the PLP-bound lysine, generating a substrate radical (Sub•) (Figs. 1 and S1). However, the crystal structure indicates a 23.4 Å distance between AdoCbl and PLP (Fig. 1), suggesting that a significant conformational change is necessary to bring the substrate and metallocofactor into proximity for the reaction. 22 This hypothesis is supported by modeling, spectroscopic studies, and DFT calculations. 22-25 Nevertheless, the precise details of this conformational shift, the mechanism by which LAM activates the Co(III)-C bond, and how the enzyme controls the highly reactive Ado • radical remain unresolved. In this study, we report cryo-electron microscopy (cryo-EM) structures of a thermophilic version of LAM in both its inactive 'Open' (substrate-free) and active 'Closed' (substrate-bound) states. Our results revealed that substrate binding induces a significant conformational shift, including a 64.1-degree rotation of the Rossmann domain. This structural rearrangement moves the AdoCbl cofactor 25.5 Å, positioning it in proximity to the substrate-bound PLP. In the activated 'Closed' state, the PLP is situated between the adenosyl and cobalamin components following the Co(III)-C bond homolysis. A hydrogen bond network involving Glu412, Lys371, and Asp299 around the AdoCbl constrains the position and conformation of the ephemeral Ado • radical, ensuring its selectivity with specific C-H bonds on the substrate. This precise control over the highly reactive Ado • radical is crucial for preventing side reactions that could compromise the enzyme's catalytic efficiency and structural integrity. Our work highlights a general principle in metalloenzymes: substrate binding triggers metallocofactor activation through conformational rearrangement, which not only activates the Co(III)-C bond but also creates a highly selective environment that confines the Ado • radical to its intended targets. This regulatory strategy parallels those observed in other metalloenzymes, such as cytochrome P450s and non-heme iron proteins, where substrate binding initiates the formation of highly reactive Fe(IV)=O species. 26,27 The intricate interplay of protein conformational changes, Co(III)-C activation and subsequent substrate reaction demonstrated in this paper highlights the sophistication, efficiency, and precision of nature's catalytic machinery. Results Expression and Activity of Thermoanaerobacter tengcongensis LAM (TtLAM). The LAM variant we selected is an uncharacterized thermophilic variant from Thermoanaerobacter tengcongensis (Tt), a microorganism originating from the hot springs in Tengchong, China.28 This microbe thrives optimally at 75 °C across a wide pH range (5.5 - 9.0), indicating a high degree of thermostability under diverse reaction conditions. Utilizing this thermophilic version of LAM minimizes the risk of denaturation or aggregation during sample preparation and imaging. Compared to other LAMs, the thermostability of TtLAMs reduces the likelihood of freezing artifacts during vitrification and enhances the preservation of its functional state in cryo-EM, allowing for more accurate insights into its mechanisms and interactions.29 The TtLAM gene was cloned into the pCOLADuet-1 vector to co-express both the α and β subunits in E. coli.30 The co-expression of both subunits promotes the in vivo formation of the tetramer, and the N-terminal His-tags on the α subunits enabled the one-step purification of the intact apoenzyme via Ni-NTA affinity chromatography, followed by anion exchange chromatography.21 The presence of both subunits was confirmed by SDS-PAGE (Fig. S2). After incubating the apoenzyme with excess PLP and AdoCbl, unbound cofactors were removed by a PD-10 desalting column. The incorporation of both cofactors in holo-TtLAM was verified by UV-Vis spectroscopy (Fig. S3).31 The catalytic efficiency of TtLAM in converting lysine to 2,5-diaminohexanoic acid (2,5-DAH) was assessed using Michaelis–Menten kinetics. At 30°C and 60°C, the enzyme exhibited a kcat of 0.67 ± 0.02 s⁻¹ and 8.2 ± 0.3 s⁻¹, respectively, with Km of 21.5 ± 1.0 mM and 24.5 ± 3.0 mM, (Fig. S4), consistent with its thermophilic origin. The product 2,5-diaminohexanoic acid (2,5-DAH) was isolated using silica column chromatography, and its identity and purity were confirmed via 1H-NMR and 13C-NMR (Figs. S5-6). Cryo-EM Structure of Holo TtLAM in the ‘Open’ state. The x-ray crystal structure of CsLAM reveals a 23.4 Å distance between AdoCbl and PLP (Fig. 1),22 leading to the hypothesis that this separation of the two cofactors is a specific design to prevent harmful radical damage at the substrate-binding site in the enzyme’s resting state.3 Alternatively, the distance might be an artifact of crystal packing in the solid state, as crystal packing can influence the positioning of protein domains, especially in flexible, multidomain proteins, potentially resulting in non-physiological conformations.32,33 Cryo-EM has emerged as a powerful technique to study proteins in more native-like conformations in solution, capturing snapshots of enzymes in action and revealing conformational changes in multidomain proteins.34,35 This technique offers a new approach for investigating LAM, allowing us to resolve its solution structures in various states, which can clarify whether the distant positions of the cofactors represent the enzyme’s native state and provide structural insight into its catalytic process in the solution phase. The Cryo-EM structure of substrate-free holo TtLAM (‘Open’ state) was solved at 3.1 Å resolution (Fig. 2). The overall structure of TtLAM forms an α2β2 tetramer, consistent with the x-ray structure of CsLAM.22 AdoCbl is bound to the Rossmann subdomain in the β subunit of TtLAM through His133β, which coordinates to the cobalt center as a axial ligand in a “base-off/His-on” conformation (Fig. 2b, left inset). The DMB moiety, replaced by His in this configuration, resides in the hydrophobic pocket of the Rossmann domain, where Ser187β forms a strong hydrogen bond with DMB at a distance of 2.6 Å. The PLP cofactor is covalently attached to the protein through an imine bond with Lys144β from the Rossmann domain of the β subunit, while the PLP itself is located in the TIM barrel of the α subunit, where its pyridine ring engages in π-π stacking with Tyr237α and Tyr264α (Fig. 2b, right inset). Additionally, the phosphate group of PLP interacts electrostatically with Arg185α and Arg269α and forms a hydrogen bond with Ser190α. Notably, in the cryo-EM structure of TtLAM, the AdoCbl and PLP cofactors are also separated by approximately 25 Å, a distance similar to that observed in the CsLAM x-ray structure. This observation confirmed that the cofactor separation is a natural feature of the enzyme, not a crystal packing artifact. As previously proposed, this separation mandates a significant conformational change during catalysis to bring the AdoCbl cofactor to the substrate bound PLP to enable HAA from the substrate by the highly reactive Ado• radical. Additionally, the cryo-EM structure revealed a previously unresolved loop connecting the Rossmann domain to the non-adjacent dimerization domain in the β subunit (Fig. 2c). Structurally, the flexible loop can serve as the hinge for Rossmann domain alternation. This discovery provides a revised structural feature of LAM compared to the earlier x-ray crystal structure.22 EPR Characterization of Holo TtLAM in ‘Close’ State. To capture TtLAM in its 'Closed' form, an inhibitor is required to initiate the conformational shift while simultaneously blocking the completion of the catalytic cycle. Mechanistically, after Co(III)-C bond homolysis, HAA occurs between the Ado• radical and the PLP-bound substrate, generating the carbon-centered Sub• radical (Fig. 1). A sulfur atom adjacent to the carbon radical is known to stabilize the radical through hyperconjugation and electron delocalization. Therefore, we used 4-thia-L-lysine as the inhibitor, in which the C4 of lysine is replaced by a sulfur atom adjacent to the carbon radical at the C5 position. This inhibitor is known for Porphyromonas gingivalis LAM (PgLAM), with previous EPR studies showing that it promotes Co(III)-C bond homolysis and induces conformational changes.23-25 Upon anaerobic addition of 4-thialysine to holo TtLAM, the EPR spectrum revealed the formation of a transient intermediate that persisted for up to 30 seconds (Fig. 3). This intermediate lasted longer than was observed in PgLAM,36 possibly due to the increased thermostability of the TtLAM. Low-spin, square-planar Co(II) compounds, including derivatives of coenzyme B12, typically exhibit g┴ values between 2.2-3.0 and A|| around 100 G.37-39 However, the EPR spectrum of the transient species captured showed g┴ = 2.12 and A|| = 45 G, accompanied by a distinctive 8-line hyperfine pattern corresponding to the nuclear spin (I = 7/2) of the Co atom, indicating the presence of a spin-coupled Co(II)/radical pair, a feature commonly observed in AdoCbl-dependent enzymes.40 The radical could be located either at the PLP-lysine conjugate (as a Sub• radical), or associated with the adenosyl group after Co(III)-C homolysis (as a Ado• radical) (Fig. 1). To pinpoint the radical's location, 13C-labeled ATP was reacted with hydroxocobalamin to synthesize 13C-AdoCbl following a previously reported procedure.41 The resulting 13C-AdoCbl was then incorporated into TtLAM along with PLP to reconstitute the holoenzyme. The reaction of 13C-AdoCbl LAM with 4-thialysine under the same conditions was freeze-quenched at 30 seconds, and the EPR spectrum revealed no significant hyperfine changes in the 13C-labeled samples compared to those with natural isotope abundance samples (Fig. 3). This absence of hyperfine changes confirmed that the radical is located at the PLP-substrate adduct rather than at the C5' position of adenosine. In PgLAM, 13C-labeled 4-thia-L-lysine was used to probe the radical location, and the radical captured was located at the PLP-substrate adduct.36 Our results aligned with the reported observations in a different LAM and excluded the possibility of Ado• radical as a co-existing reactive species in the transient intermediate. The Co(II)/ Sub• radical pair interacts through dipole-dipole and exchange interactions, both of which are influenced by orbital overlap and spin polarization effects.40 The dipole-dipole interaction is particularly valuable for structural analysis because it follows a 1/r³ dependence on the distance between the Co(II) and Sub• radical pair. For distances greater than 4 Å, this interaction breaks the degeneracy of the spin states in the absence of an external magnetic field, resulting in zero-field splitting, which is described by axial D and rhombic E terms. The D term is distance-dependent and is described by the equation D = D₀/r³, where r (Å) is the inter-spin distance, and D₀ = 2.785 × 10⁴ Gų.40 By determining the D term, the distance between the Co(II) and Sub• radical pair can be calculated. The EPR spectrum of Co(II)/ Sub• radical pair was simulated as an axial low-spin Co(II) species (g┴Co = 2.27, g||Co = 2.0, A┴Co = 20 G, A||Co = 100 G),37 strongly coupled with an isotropic carbon radical species (giso,C = 2.003), with zero-field splitting parameters D = -152.4 G (Fig. 3). This corresponded to a calculated distance of 5.7 Å between the Co(II) and Sub• radical. To further explore how distance affects the EPR spectra, we simulated the spectra by varying the distance between the Co(II) and Sub• radical (Fig. S7). For distances less than 7 Å, no significant changes were observed in the simulated EPR spectra. This is consistent with the observed g┴ values of 2.12 for the Co(II)/ Sub• radical pair, which is smaller than the typical g┴ values of 2.2-3.0 for low-spin, square-planar Co(II) compounds. The decrease in g┴ was simulated to result from exchange interactions between the two spin systems at distances below 7 Å, leading to g-value averaging and eventually converging to a triplet state.40 Distances longer than 7 Å led to significant changes in the simulated EPR spectra due to the weakening of both dipole-dipole and exchange interactions as the spatial separation increases. As shown in Fig. 2, the AdoCbl and PLP cofactors are separated by a distance of approximately 25 Å. The EPR calculated distance of less than 7 Å between the Co(II) and Sub• radical suggested a conformational change upon substrate binding, which brings the AdoCbl cofactor and the PLP-Sub adduct closer. Therefore, spectroscopic evidence indicated that the ‘Closed’ state in LAM involves not only a TIM barrel conformational change similar to MCM,14 but also a more substantial movement that reduces spatial distance between the two cofactors from approximately 25 Å to less than 7 Å. Cryo-EM Structure of Holo TtLAM in ‘Close’ State. With the timing of the transient intermediate identified by EPR spectroscopy, we captured the ‘Closed’ state of TtLAM by freeze-quenching the mixture of holo TtLAM and 4-thia-L-lysine in liquid ethane at the 30 s timepoint. The cryo-EM structure of TtLAM in the ‘Closed’ state was solved at 3.0 Å resolution (Fig. 4). In one β subunit (β'), the dimerization subdomain is well-resolved, while the Rossman domain is invisible, possibly because the mixture was quenched during the dynamic motion of the subunit during catalysis. In the other β subunit, both the dimerization and Rossman domains were fully resolved (Fig. 4a). Superimposed structures of TtLAM in the ‘Open’ and ‘Closed’ conformations revealed a substantial 64.1° rotation of the Rossman domain, while the dimerization domains and α subunits remained static (Fig. 4c). Since the AdoCbl cofactor is anchored to the Rossman domain, this rotation shifted the cofactor by an unprecedented 25.5 Å, bringing it closer to the PLP-substrate adduct (Fig. 4b). The active site of TtLAM in the ‘Closed’ state is shown in Fig. 5. The cobalamin remains bound to the Rossmann domain in a “base-off/His-on” conformation, with His133β serving as the lower axial ligand to the cobalt center (Fig. 5a). The position of the PLP cofactor is unchanged between the ‘Open’ and ‘Closed’ states. In both conformations, PLP is clamped between Tyr237α and Tyr264α through π-π stacking interactions between the pyridine ring of PLP and the tyrosine rings on either side. The phosphate group of PLP is locked by electrostatic interactions with Arg185α and Arg269α. The key difference in the PLP cofactor between the ‘Open’ and ‘Closed’ forms lies in its interaction with Lys144β. In the ‘Open’ state, PLP is covalently bound to Lys144β via an imine bond (Fig. 2b). However, in the ‘Closed’ state, Lys144β is replaced by 4-thia-L-lysine through a transimination reaction. Therefore, in ‘Closed’ state, there is no covalent linkage between PLP and the Rossmann domain. This substitution frees Lys144β, allowing rotation of the Rossmann domain independent from PLP. Notably, the distance between Co(II) and the C5 position of 4-thia-L-lysine is 6.3 Å (Fig. 5a), consistent with the distance of < 7 Å between Co(II) and the Sub• radical as measured by EPR spectroscopy (Fig. 3). Overall, our cryo-EM structure provides direct structural information to confirm the long-existing hypothesis of Rossmann domain rotation in LAM activation. Another key question in the LAM mechanism is how the enzyme controls the highly reactive Ado• radical formed upon Co(III)–C bond homolysis. The bond dissociation energy (BDE) for the adenosine C5′–H bond is estimated to be between 94 and 101 kcal/mol.42 The highly reactive carbon-centered Ado• radical could potentially react with various C-H bonds or other functional groups within the enzyme's substrate binding site, leading to side reactions that might compromise the integrity of the catalytic site. The cryo-EM structure of the ‘Closed’ state revealed that the cleaved adenosine is tightly restricted in the active site through hydrogen bonds with Lys371α and Asp299α (Fig. 5b), preventing rotation around the C–N single bond between adenine and ribose and consequently any conformation or position change of the ribose group. Additionally, with the PLP locked in place, the substrate lysine is positioned between the cobalamin and adenosine. The C5' of adenosine is precisely aligned with the C5 and C6 positions of 4-thia-lysine, maintaining distances of 4.7 Å and 5.1 Å, respectively (Fig. 5c). This controlled spatial arrangement ensures that the Ado• radical remains properly oriented toward the substrate, preventing unwanted side reactions and protecting the catalytic site. Moreover, the alignment ensures the correct shift of the amino group between C5 and C6 to produce the desired product, illustrating how TtLAM controls the selectivity of the isomerization reaction. Systematic Mutation of the Hydrogen Bond Network Around Adenosine. In addition to the precise positioning of adenosine by Asp299α, Glu412α, and Lys371α in the ‘Closed’ form (Fig. 5), the hydrogen bond network may also play an integral role in facilitating the Co(III)-C homolysis. To test this hypothesis, we systematically mutated the residues involved in the hydrogen bond network and monitored Co(III)-C bond homolysis using EPR spectroscopy. In the E412Q mutant, the carboxylic side chain of Glu was replaced with the amide in Gln, which could weaken H-bond or alter local electric field and consequently affect both the adenosine and the periphery of the corrin ring. The mutant was loaded with PLP and AdoCbl and reacted with 4-thia-L-lysine. The reaction mixture was quenched at 30 s, and the corresponding ERP spectra was silent, indicating that the subtle change induced by the mutation prevented the homolysis of the Co(III)-C bond and the formation of the Co(II)/ Sub• radical pair (Fig. 3). Similarly, the reaction mixture of the K371Q mutant was also EPR silent (Fig. 3). The reaction mixture of D299N mutant, which was designed similar to E412Q, produced radical pair EPR signals similar to the WT but with much lower intensity (Fig. 3), indicating that Co(III)-homolysis occurred but with reduced yield. These mutation studies confirmed the critical role of the hydrogen bond network and local electric field in the active site in facilitating the Co(III)-C bond cleavage. Discussion Substrate binding can trigger conformational changes in proteins, enabling or enhancing their catalytic activity. This mechanism is widely employed by nature in many enzymatic processes and is fundamental to a range of biological functions. Most enzymes undergo only minute conformational changes, typically involving small shifts in the geometry of catalytic residues, usually less than 1 Å. 43 In some enzymes, more significant movements of the binding residues occur, especially when these residues are located on the surface loops. 44 Domain alternation refers to larger-scale conformational changes, where different domains of an enzyme shift between distinct positions or orientations. 45 Although rare in metalloproteins, domain alternation also can play a crucial role in regulating their catalytic activity. For example, in methionine synthase (MetH), movement of the cobalamin-binding cap domain creates a new interface between cobalamin and the S-adenosylmethionine (SAM)-binding domain, facilitating the transfer of a methyl group from SAM to cobalamin. 46,47 Similarly, in ribonucleotide reductase (RNR), substrate binding to the α subunit induces domain rearrangements that bring the α and β subunits into close proximity, enabling the 32 Å-long radical transfer pathway essential for RNR activity. 48 Our findings showcased domain alternation as a mechanism for activating the metallocofactor in Tt LAM, as supported by cryo-EM structures and ERP spectroscopy. Simply binding the cofactor AdoCbl to the protein scaffold does not activate the Co(III)-C bond. 11 To cleave the Co(III)-C bond of 30 kcal dissociation energy and achieve the enzymatic turnover rate, the activation process must reduce the energy required for bond cleavage by 17 kcal/mol. As demonstrated by x-ray and our cryo-EM structures, in the ‘Open’ state, PLP is located in the TIM barrel domain (α subunit) and is also covalently linked with Lys144β of the Rossmann domain within the β subunit. (Fig. 2b). Therefore, PLP acts as an anchor, connecting the TIM barrel and Rossmann domains and enforcing an unconventional orientation of the Rossmann domain. The cryo-EM structure of the ‘Closed’ state revealed that, upon substrate binding, transimination frees Lys144β, dismantling the PLP-mediated anchoring between the Rossmann and TIM barrel domains. This release triggers a substantial conformational shift, including a 64.1-degree rotation of the Rossmann domain, which moves the AdoCbl cofactor by 25.5 Å (Fig. 4c) into a new binding site. In the new site, the dissociated adenosine is stabilized by a hydrogen bond network involving Asp299α, Glu412α, and Lys371α from the TIM barrel. (Fig. 5). These hydrogen bonds, all located on one side of adenosine opposite the cobalamin, not only restrict the adenosine’s position after dissociation, but may also exert a "pull" effect on the adenosyl group even before the Co(III)-C bond cleavage, shifting it away from the cobalamin and weakening the Co(III)-C bond, thereby decreasing the bond dissociation energy and promoting its homolytic cleavage. This substrate-controlled metallocofactor activation mirrors mechanisms observed in heme and non-heme iron proteins. For instance, in cytochrome P450 enzymes, substrate binding forces the heme iron to transition from a six-coordinate to a five-coordinate state, resulting in a spin-state switch from low-spin to high-spin. The high-spin state facilitates electron transfer to the Fe³⁺ center to form the Fe²⁺ reduced state, which is necessary for oxygen activation and formation of the highly reactive Fe(IV)=O species. 26 Similarly, in α-ketoglutarate (α-KG)-dependent non-heme iron dioxygenases, the binding of α-KG and the substrate induces a shift from a six-coordinate to a five-coordinate iron center, creating a vacant site essential for oxygen binding and activation. 27 A similar concept applies here: without the substrate, the Co(III)-C bond will not be activated to generate the highly reactive Ado • radical. Co(III)-C bond activation only occurs in the presence of the substrate, ensuring that the generated Ado • radical can be quenched by the substrate C-H bond. However, this process is distinct in that the activation is driven by protein domain alternation and metallocofactor relocation rather than changes in the coordination of the metallocofactors. Radical-based chemistry offers exceptional reactivity but carries the inherent risk of side reactions due to the highly reactive nature of radical intermediates, which can deactivate the enzyme. This work illustrated the elegant natural design of LAM that controls radical generation through substrate binding and mitigates radical damage via binding site design. The sophisticated hydrogen-bonding network surrounding adenosine after Co(III)-C bond homolysis effectively directs the radical toward the substrate. These structures elegantly demonstrate how the protein fold harnesses radical-based chemistry, emphasizing both the precision and protective strategies employed by the enzyme. Conclusion Our investigation of Tt LAM using cryo-EM and EPR provided crucial insights into the enzyme’s activation mechanism. The cryo-EM structures experimentally demonstrated, for the first time, a 64.1-degree rotation of the Rossmann domain induced by substrate binding, which relocates the AdoCbl cofactor by 25.5 Å. This movement is essential for activating the Co(III)-C bond to form the highly reactive Ado • radical. In the ‘Closed’ state, key hydrogen-bond network involving Lys371α, Asp299α, and Glu412α around adenosine ensure the precise orientation of the Ado • radical toward the PLP-conjugated lysine, controlling HAA at the C5 or C6 positions of the substrate and preventing harmful side reactions. By employing 4-thialysine, the transient Sub • was freeze quenched, and EPR spectra confirmed the proximity of the cobalt center and the radical located on the substrate. This work highlighted the sophisticated structural strategies employed by LAM to efficiently and safely harness radical-based chemistry, offering broader implications for understanding metallocofactor activation in other radical-based enzymes. Declarations Acknowledgements We acknowledge the U.S. National Institute of General Medical Sciences (R35GM155016 to S.T.) for funding the research described in this work. We also thank Dr. Tatsuo Kurihara (Kyoto University) for generously providing the plasmid for Tt LAM expression and Dr. Jorge Escalante (University of Georgia) for the plasmid for CobA expression. Additionally, we are grateful to Purdue University's Research Instrumentation Center for access to EPR, facilitated by the Amy Instrumentation Facility, Department of Chemistry, under the supervision of Dr. Michael Everly and Dr. Aloke Bera. We also extend our thanks to the Purdue Cryo-EM Facility for providing access to the Titan Krios instrument. References Banerjee, R. 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Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases 83 , 3663-3667, (1987). Reed, G. H. & Mansoorabadi, S. O. The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes. Curr. Opin. Struct. Biol. 13 , 716-721, (2003). Tang, K.-H., Chang, C. H. & Frey, P. A. Electron Transfer in the Substrate-Dependent Suicide Inactivation of Lysine 5,6-Aminomutase. Biochemistry 40 , 5190-5199, (2001). Luo, Y.-R. Handbook of bond dissociation energies in organic compounds . (CRC Press, 2003). Gutteridge, A. & Thornton, J. Conformational change in substrate binding, catalysis and product release: an open and shut case? FEBS Lett. 567 , 67-73, (2004). Henzler-Wildman, K. A. et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450 , 913-916, (2007). Gulick, A. M. Conformational Dynamics in the Acyl-CoA Synthetases, Adenylation Domains of Non-ribosomal Peptide Synthetases, and Firefly Luciferase. ACS Chemical Biology 4 , 811-827, (2009). Bandarian, V. et al. Domain alternation switches B12-dependent methionine synthase to the activation conformation. Nat. Struct. Biol. 9 , 53-56, (2002). Watkins, Maxwell B., Wang, H., Burnim, A. & Ando, N. Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy. Proc. Natl. Acad. Sci. U.S.A. 120 , e2302531120, (2023). Kang, G., Taguchi, A. T., Stubbe, J. & Drennan, C. L. Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex. Science 368 , 424-427, (2020). Additional Declarations There is NO Competing Interest. 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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-5313240","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":369461541,"identity":"73265a1a-1f2c-47d7-8c8b-8d63cec011df","order_by":0,"name":"Shiliang 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16:01:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5313240/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5313240/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68234392,"identity":"e0fb753a-120e-4597-8ee3-f8594d3b6711","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and mechanistic considerations for 5,6-lysine aminomutase.\u003c/strong\u003e a) Structures of adenosylcobalamin (AdoCbl) and lysine-bound pyridoxal phosphate (PLP). b) Free AdoCbl has a bond dissociation energy (BDE) of 30 kcal/mol, leading to a predicted bond dissociation rate (BDR) of 10\u003csup\u003e-9\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. When in the protein scaffold, the reactions of AdoCbl dependent proteins occurs with turnover rates of 10 to 100 s\u003csup\u003e-1\u003c/sup\u003e. These rates suggest a weaking of the Co(III)-C bond in the AdoCbl cofactor to 13 kcal/mol when in the protein scaffold. c) Relative positions of the PLP and AdoCbl cofactors within the previously solved crystal structure are separated by 23.4 Å. Proposed mechanisms include cleavage of the Co(III)-C bond in the presence of substrate to form Co(II)-Cbl and the Ado\u003csup\u003e•\u003c/sup\u003e radical. The highly reactive Ado\u003csup\u003e•\u003c/sup\u003e radical is known to abstract a hydrogen atom from the PLP bound substrate leading to the formation of Sub\u003csup\u003e•\u003c/sup\u003e. This mechanistic insight highlights the necessity for the AdoCbl cofactor to be in close proximity to the PLP cofactor, emphasizing the need for further structural understanding of the active form of 5,6-LAM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/03b9a788ab8473c0063fe2dd.png"},{"id":68234395,"identity":"f534a15f-b683-459d-8985-f95a9072316a","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":367417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLAM in ‘Open’ state with 3.1 Å resolution. \u003c/strong\u003e(a) EM density maps showing the α\u003csub\u003e2\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003e heterotetrameric structure of \u003cem\u003eTt\u003c/em\u003eLAM. The α subunits (TIM barrel) are shown in magenta and cyan, while the β subunits (Rossman and dimerization domains) are depicted in dark blue and green. (b) EM density corresponding to the AdoCbl and PLP cofactors, revealing significant spatial separation characteristic of the enzyme's ‘Open’ form. The AdoCbl (left inset) is coordinated by His133 from the β subunit in a \"base-off/His-on\" conformation, with the DMB moiety anchored in the Rossmann subdomain of the β subunit. The PLP (right inset) is covalently bound to Lys144 of the β subunit, forming an internal aldimine and interacting with two tyrosine residues (Tyr237α and Tyr264α) via π-π stacking. (c) Inset highlighting a previously unresolved loop connecting the Rossmann fold to the non-adjacent dimerization domain in the β subunit. The two α subunits are represented in gray for clarity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/2787f7406741ba91eda62822.png"},{"id":68234394,"identity":"db0e62c9-fa4e-4c77-8864-d744959cfe54","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPR spectra of the transient biradical intermediate. \u003c/strong\u003eThe transient intermediate was generated by anaerobic addition of 4-thialysine to holo \u003cem\u003eTt\u003c/em\u003eLAM while stirring. The mixture was incubated for 30 seconds before being frozen in liquid nitrogen. The EPR spectrum of the transient intermediate of WT TtLAM is shown in red, with its simulation in black. The spectrum was simulated as a low-spin Co(II) (S = 1/2) species strongly coupled with an isotropic carbon radical species. The simulation parameters are: g\u003csub\u003e┴Co\u003c/sub\u003e = 2.27, g\u003csub\u003e||Co\u003c/sub\u003e = 2.0, A\u003csub\u003e┴Co\u003c/sub\u003e = 20 G, A\u003csub\u003e||Co\u003c/sub\u003e = 100 G, g\u003csub\u003eiso,C\u003c/sub\u003e = 2.003, zero-field splitting parameters D = -152.4 G, E = 21.7 G, exchange coupling constant J = 3700 G, and Euler angles of the g frame of Co(II) aligning with the g frame of the PLP-substrate radical = [27°, 24°, -10°]. The EPR spectrum of the \u003csup\u003e13\u003c/sup\u003eC-labeled adenosine is shown in blue. The lack of hyperfine changes in the \u003csup\u003e13\u003c/sup\u003eC-labeled samples compared to natural abundance samples confirms that the radical is located at the PLP-substrate adduct, rather than on adenosine. EPR spectra of \u003cem\u003eTt\u003c/em\u003eLAM mutants are shown for D299N (green), E412Q (purple), and K371Q (pink).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/134f3d205459dd4954da1475.png"},{"id":68234393,"identity":"ed886e83-5617-4bfc-b676-7c1319e0569d","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":255335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLAM in ‘Closed’ state at 3.0 Å resolution. \u003c/strong\u003e(a) EM density maps showing the α\u003csub\u003e2\u003c/sub\u003eβ\u003csub\u003e2\u003c/sub\u003e heterotetrameric structure of \u003cem\u003eTt\u003c/em\u003eLAM in the ‘Closed’ conformation. The α subunits are colored in magenta and cyan, while the β subunits are depicted in dark blue and green. In one β subunit (β'), the dimerization subdomain is resolved, while the Rossman subdomain is not visible due to its motion during catalysis. Both the dimerization and Rossman subdomains are well-resolved in the other β subunit. (b) Overlay of the EM density map and the fitted structure of \u003cem\u003eTt\u003c/em\u003eLAM, highlighting the catalytic sites with the AdoCbl and PLP complex. (c) Superimposed structures of \u003cem\u003eTt\u003c/em\u003eLAM in ‘Open’ and ‘Closed’ conformations. The ‘Open’ conformation is shown with light pink α subunits and light green β subunits, while the 'Closed' conformation is represented with magenta α subunits and dark green β subunits. The AdoCbl cofactor is depicted in light blue for the ‘Open’ state and blue for the ‘Closed’ state. The dashed line indicates the distance between the Co atoms in the ‘Open’ and ‘Closed’ conformations. An arrow highlights the rotation of the Rossman subdomain upon substrate binding.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/265550b2f9abb9b663607adc.png"},{"id":68234397,"identity":"253d0575-4550-4b7b-a8fe-56dd00f08e0a","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":363712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActive site of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eLAM in ‘Closed’ state. \u003c/strong\u003e(a) A close-up view of the AdoCbl/PLP-substrate active site in the 'Closed' conformation. The PLP-substrate complex is depicted in green, the 5'-deoxyadenosyl group in orange, and the cobalamin cofactor in cyan. Key amino acid residues interacting with the homolyzed AdoCbl cofactor and the PLP-substrate adduct are labeled and highlighted in magenta. Dashed black lines represent hydrogen bonds and interactions, with distances between atoms that are critical for catalysis. The mesh shows the EM density. (b) A detailed view of the adenosine moiety formed from the homolysis of the Co(III)-C bond. Hydrogen-bonding interactions between Lys371α and Asp299α are displayed, which restrict the movement of the highly reactive Ado\u003csup\u003e•\u003c/sup\u003e, preventing unwanted side reactions. (c) A close-up view showing the spatial relationship between adenosine and the PLP-substrate adduct. The distances between C5’ of Ado and C5/C6 of the substrate are shown, illustrating how hydrogen atom abstraction can occur, leading to the formation of a substrate-based radical at either the C5 or C6 positions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/446cec6a6970e4f090123d4e.png"},{"id":70044809,"identity":"69dffaa3-02c8-42f9-b6d5-3e86f5dcfb8f","added_by":"auto","created_at":"2024-11-27 19:03:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1823441,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/62ce64cc-a709-46d1-aeab-4f24a2a09648.pdf"},{"id":68234398,"identity":"c76788c1-9387-4675-a631-3cd4b083ca14","added_by":"auto","created_at":"2024-11-05 06:44:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1569982,"visible":true,"origin":"","legend":"","description":"","filename":"LAMSI20241022.docx","url":"https://assets-eu.researchsquare.com/files/rs-5313240/v1/cfc6535225be6651f4e41f9a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Catalysis in Motion: Large-Scale Domain Alternation Enables Co(III)-C Bond Homolysis in Lysine 5,6-Aminomutase","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoenzyme B\u003csub\u003e12\u003c/sub\u003e, or 5\u0026rsquo;-deoxyadenosylcobalamin (AdoCbl), is a biologically active form of vitamin B\u003csub\u003e12\u003c/sub\u003e and one of the most complex metalloprotein cofactors.\u003csup\u003e1,2\u003c/sup\u003e The organometallic complex features cobalt coordinated by a corrin ring, which is functionalized with several substituents, including a dimethylbenzimidazole (DMB) tail that serves as an axial ligand to the cobalt center. In AdoCbl-dependent enzymes, the axial DMB is often replaced by a histidine residue, which not only coordinates the cobalt but also anchors the cofactor to the protein. Members of the AdoCbl enzyme family share common structural and functional features, including a triose-phosphate isomerase (TIM) barrel domain for substrate binding and a Rossmann domain for AdoCbl binding.\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdoCbl-dependent enzymes play crucial roles in several fermentation pathways, particularly in anaerobic organisms.\u003csup\u003e2\u003c/sup\u003e A notable example is the Wood-Ljungdahl pathway, where AdoCbl-dependent enzymes facilitate the conversion of CO\u003csub\u003e2\u003c/sub\u003e into acetyl-CoA, a key coenzyme involved in many metabolic processes. This pathway holds significant potential for biofuel production and carbon sequestration.\u003csup\u003e4\u003c/sup\u003e Additionally, AdoCbl-dependent enzymes have important implications in medical fields. Alterations that impair these enzymes\u0026apos; functionality can lead to severe genetic disorders. For instance, a mutation in the gene encoding methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, a condition characterized by dangerous acid buildup in the body.\u003csup\u003e5\u003c/sup\u003e Furthermore, AdoCbl-dependent enzymes are potential targets for cancer therapies. Cobalamin is an essential co-factor for methionine synthase, a key enzyme in DNA synthesis and repair. Disrupting this process could potentially inhibit cancer cell proliferation.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAdoCbl-dependent enzymes catalyze radical-mediated reactions that initiate with the homolytic cleavage of the Co(III)\u0026minus;C bond in the AdoCbl cofactor, generating an Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical, which then abstracts a hydrogen atom from the substrate.\u003csup\u003e1,3,7\u003c/sup\u003e Outside the protein environment, the Co(III)\u0026minus;C bond has a dissociation energy of approximately 30 kcal/mol, corresponding to an estimated bond dissociation rate constant of ~3.8 x 10⁻⁹ s⁻\u0026sup1;.\u003csup\u003e1\u003c/sup\u003e However, the catalytic rate constants of AdoCbl-dependent enzymes are typically in the range from 10 to 100 s⁻\u0026sup1;, which implies a reduced Co(III)-C bond dissociation energy around 13 kcal/mol (Fig. 1). To achieve this remarkable rate enhancement over 10\u003csup\u003e9\u003c/sup\u003e fold, the enzymes must destabilize the Co(III)-C bond by about 17 kcal/mol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVarious mechanisms have been proposed to explain the activation of the Co(III)\u0026minus;C bond in the enzymatic environment. Early studies indicated that a trans effect of the histidine, as an axial ligand to the cobalt center, could weaken the Co-C bond.\u003csup\u003e8-10\u003c/sup\u003e However, resonance Raman (rR) measurements showed only a slight shift in the Co(III)-C bond vibration in MCM, from 424 cm⁻\u0026sup1; in the enzyme-bound form to 420 cm⁻\u0026sup1; in the free cofactor form, indicating that histidine replacement of the DMB ligand and protein binding only weakens the Co(III)-C bond by about 0.5 kcal/mol.\u003csup\u003e11\u003c/sup\u003e Distortion of the corrin ring has been reported to destabilize the Co-C bond, but primarily in model coplexes.\u003csup\u003e11-13\u003c/sup\u003e In MCM, crystallography studies revealed that in the absence of substrate, the TIM-barrel domain remains open. Upon substrate binding, the barrel closes, displacing the adenosyl group of AdoCbl and facilitating the formation of the Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical. These conformational changes have been calculated to lower the barrier for Co(III)-C bond homolysis by approximately 7 kcal/mol.\u003csup\u003e14,15\u003c/sup\u003e Additionally, electrostatic interactions between the ribose of the adenosyl group and amino acid residues in the MCM\u0026apos;s active site are critical for catalysis. Removal of the 2\u0026rsquo;-OH from the natural cofactor results in a rate decrease of up to two orders of magnitude.\u003csup\u003e15-17\u003c/sup\u003e A tyrosine (Tyr) residue near the AdoCbl cofactor in MCM \u0026nbsp;has been calculated to lower the reaction barrier by 1 kcal/mol via a hydrogen bond network formed between its \u0026minus;OH group, the carboxylate group of the substrate, and a side chain of AdoCbl.\u003csup\u003e18\u003c/sup\u003e This interaction promotes proton-coupled electron transfer (PCET) between the Tyr and AdoCbl, generating a [AdoCbl]\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/Tyr\u003csup\u003e\u0026bull;\u003c/sup\u003e diradical state.\u003csup\u003e19,20\u003c/sup\u003e While these studies have provided valuable insights into the Co(III)-C homolysis in MCM, the mechanisms driving the 10⁹-fold rate acceleration of Co(III)\u0026minus;C bond homolysis in other AdoCbl-dependent enzymes remain largely unexplained.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003eBeyond Co(III)-C activation, AdoCbl-dependent catalysis also features highly controlled radical chemistry. After the cleavage of the Co(III)-C bond in AdoCbl, the Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical must be positioned to abstract a hydrogen atom from the substrate. This highly reactive, carbon-centered radical is a key intermediate in AdoCbl-dependent enzymatic reactions. In principle, this radical could react with C-H bonds or other functional groups within the substrate-binding site, potentially leading to side reactions that could compromise the integrity of the catalytic site. However, no such side reactions have been observed in AdoCbl-dependent enzymes. How do these enzymes protect the highly reactive Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical during catalysis? How do they ensure reaction selectivity to guarantee the formation of the intended products? What precise structural changes occur during these enzymatic processes? Answering these questions will not only deepen our understanding of the structure-function relationships and mechanisms of AdoCbl-dependent mutases but also enhance their potential as therapeutic targets for various cancers.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eLysine 5,6-aminomutase (LAM) is a member of a subfamily of AdoCbl-dependent enzymes that utilizes an additional cofactor, pyridoxal 5\u0026prime;-phosphate (PLP), for catalysis. LAM catalyzes the reversible shift of an amino group between the C5 and C6 positions of D-lysine and L-\u0026beta;-lysine.\u003csup\u003e21\u003c/sup\u003e The x-ray crystal structure of LAM from \u003cem\u003eClostridium sticklandii\u003c/em\u003e (\u003cem\u003eCs\u003c/em\u003eLAM) reveals that it functions as an \u0026alpha;\u003csub\u003e2\u003c/sub\u003e\u0026beta;\u003csub\u003e2\u003c/sub\u003e tetramer.\u003csup\u003e22\u003c/sup\u003e The enzyme employs TIM barrel architectures for PLP and substrate binding, while AdoCbl is coordinated through Rossmann domains. Although PLP resides within the TIM barrel, lysine 144, which is covalently linked to PLP, is from the Rossmann domain. Upon substrate binding to PLP and Co(III)-C bond homolysis, hydrogen atom abstraction (HAA) occurs between the Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical and the PLP-bound lysine, generating a substrate radical (Sub\u0026bull;) (Figs. 1 and S1). However, the crystal structure indicates a 23.4 \u0026Aring; distance between AdoCbl and PLP (Fig. 1), suggesting that a significant conformational change is necessary to bring the substrate and metallocofactor into proximity for the reaction.\u003csup\u003e22\u003c/sup\u003e This hypothesis is supported by modeling, spectroscopic studies, and DFT calculations.\u003csup\u003e22-25\u003c/sup\u003e Nevertheless, the precise details of this conformational shift, the mechanism by which LAM activates the Co(III)-C bond, and how the enzyme controls the highly reactive Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical remain unresolved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we report cryo-electron microscopy (cryo-EM) structures of a thermophilic version of LAM in both its inactive \u0026apos;Open\u0026apos; (substrate-free) and active \u0026apos;Closed\u0026apos; (substrate-bound) states. Our results revealed that substrate binding induces a significant conformational shift, including a 64.1-degree rotation of the Rossmann domain. This structural rearrangement moves the AdoCbl cofactor 25.5 \u0026Aring;, positioning it in proximity to the substrate-bound PLP. In the activated \u0026apos;Closed\u0026apos; state, the PLP is situated between the adenosyl and cobalamin components following the Co(III)-C bond homolysis. A hydrogen bond network involving Glu412, Lys371, and Asp299 around the AdoCbl constrains the position and conformation of the ephemeral Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical, ensuring its selectivity with specific C-H bonds on the substrate. This precise control over the highly reactive Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical is crucial for preventing side reactions that could compromise the enzyme\u0026apos;s catalytic efficiency and structural integrity. Our work highlights a general principle in metalloenzymes: substrate binding triggers metallocofactor activation through conformational rearrangement, which not only activates the Co(III)-C bond but also creates a highly selective environment that confines the Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical to its intended targets. This regulatory strategy parallels those observed in other metalloenzymes, such as cytochrome P450s and non-heme iron proteins, where substrate binding initiates the formation of highly reactive Fe(IV)=O species.\u003csup\u003e26,27\u003c/sup\u003e The intricate interplay of protein conformational changes, Co(III)-C activation and subsequent substrate reaction demonstrated in this paper highlights the sophistication, efficiency, and precision of nature\u0026apos;s catalytic machinery.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eExpression and Activity of Thermoanaerobacter tengcongensis LAM (TtLAM). The LAM variant we selected is an uncharacterized thermophilic variant from Thermoanaerobacter tengcongensis (Tt), a microorganism originating from the hot springs in Tengchong, China.28 This microbe thrives optimally at 75 \u0026deg;C across a wide pH range (5.5 - 9.0), indicating a high degree of thermostability under diverse reaction conditions. Utilizing this thermophilic version of LAM minimizes the risk of denaturation or aggregation during sample preparation and imaging. Compared to other LAMs, the thermostability of TtLAMs reduces the likelihood of freezing artifacts during vitrification and enhances the preservation of its functional state in cryo-EM, allowing for more accurate insights into its mechanisms and interactions.29\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe TtLAM gene was cloned into the pCOLADuet-1 vector to co-express both the \u0026alpha; and \u0026beta; subunits in E. coli.30 The co-expression of both subunits promotes the in vivo formation of the tetramer, and the N-terminal His-tags on the \u0026alpha; subunits enabled the one-step purification of the intact apoenzyme via Ni-NTA affinity chromatography, followed by anion exchange chromatography.21 The presence of both subunits was confirmed by SDS-PAGE (Fig. S2). After incubating the apoenzyme with excess PLP and AdoCbl, unbound cofactors were removed by a PD-10 desalting column. The incorporation of both cofactors in holo-TtLAM was verified by UV-Vis spectroscopy \u0026nbsp;(Fig. S3).31\u003c/p\u003e\n\u003cp\u003eThe catalytic efficiency of TtLAM in converting lysine to 2,5-diaminohexanoic acid (2,5-DAH) was assessed using Michaelis\u0026ndash;Menten kinetics. \u0026nbsp;At 30\u0026deg;C and 60\u0026deg;C, the enzyme exhibited a kcat of 0.67 \u0026plusmn; 0.02 s⁻\u0026sup1; and 8.2 \u0026plusmn; 0.3 s⁻\u0026sup1;, respectively, with Km of 21.5 \u0026plusmn; 1.0 mM and 24.5 \u0026plusmn; 3.0 mM, (Fig. S4), consistent with its thermophilic origin. The product 2,5-diaminohexanoic acid (2,5-DAH) was isolated using silica column chromatography, and its identity and purity were confirmed via 1H-NMR and 13C-NMR (Figs. S5-6).\u003c/p\u003e\n\u003cp\u003eCryo-EM Structure of Holo TtLAM in the \u0026lsquo;Open\u0026rsquo; state. The x-ray crystal structure of CsLAM reveals a 23.4 \u0026Aring; distance between AdoCbl and PLP (Fig. 1),22 leading to the hypothesis that this separation of the two cofactors is a specific design to prevent harmful radical damage at the substrate-binding site in the enzyme\u0026rsquo;s resting state.3 Alternatively, the distance might be an artifact of crystal packing in the solid state, as crystal packing can influence the positioning of protein domains, especially in flexible, multidomain proteins, potentially resulting in non-physiological conformations.32,33 Cryo-EM has emerged as a powerful technique to study proteins in more native-like conformations in solution, capturing snapshots of enzymes in action and revealing conformational changes in multidomain proteins.34,35 This technique offers a new approach for investigating LAM, allowing us to resolve its solution structures in various states, which can clarify whether the distant positions of the cofactors represent the enzyme\u0026rsquo;s native state and provide structural insight into its catalytic process in the solution phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Cryo-EM structure of substrate-free holo TtLAM (\u0026lsquo;Open\u0026rsquo; state) was solved at 3.1 \u0026Aring; resolution (Fig. 2). The overall structure of TtLAM forms an \u0026alpha;2\u0026beta;2 tetramer, consistent with the x-ray structure of CsLAM.22 AdoCbl is bound to the Rossmann subdomain in the \u0026beta; subunit of TtLAM through His133\u0026beta;, which coordinates to the cobalt center as a axial ligand in a \u0026ldquo;base-off/His-on\u0026rdquo; conformation (Fig. 2b, left inset). The DMB moiety, replaced by His in this configuration, resides in the hydrophobic pocket of the Rossmann domain, where Ser187\u0026beta; forms a strong hydrogen bond with DMB at a distance of 2.6 \u0026Aring;. The PLP cofactor is covalently attached to the protein through an imine bond with Lys144\u0026beta; from the Rossmann domain of the \u0026beta; subunit, while the PLP itself is located in the TIM barrel of the \u0026alpha; subunit, where its pyridine ring engages in \u0026pi;-\u0026pi; stacking with Tyr237\u0026alpha; and Tyr264\u0026alpha; (Fig. 2b, right inset). Additionally, the phosphate group of PLP interacts electrostatically with Arg185\u0026alpha; and Arg269\u0026alpha; and forms a hydrogen bond with Ser190\u0026alpha;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, in the cryo-EM structure of TtLAM, the AdoCbl and PLP cofactors are also separated by approximately 25 \u0026Aring;, a distance similar to that observed in the CsLAM x-ray structure. This observation confirmed that the cofactor separation is a natural feature of the enzyme, not a crystal packing artifact. As previously proposed, this separation mandates a significant conformational change during catalysis to bring the AdoCbl cofactor to the substrate bound PLP to enable HAA from the substrate by the highly reactive Ado\u0026bull; radical. Additionally, the cryo-EM structure revealed a previously unresolved loop connecting the Rossmann domain to the non-adjacent dimerization domain in the \u0026beta; subunit (Fig. 2c). Structurally, the flexible loop can serve as the hinge for Rossmann domain alternation. This discovery provides a revised structural feature of LAM \u0026nbsp;compared to the earlier x-ray crystal structure.22\u003c/p\u003e\n\u003cp\u003eEPR Characterization of Holo TtLAM in \u0026lsquo;Close\u0026rsquo; State. To capture TtLAM in its \u0026apos;Closed\u0026apos; form, an inhibitor is required to initiate the conformational shift while simultaneously blocking the completion of the catalytic cycle. Mechanistically, after Co(III)-C bond homolysis, HAA occurs between the Ado\u0026bull; radical and the PLP-bound substrate, generating the carbon-centered Sub\u0026bull; radical (Fig. 1). A sulfur atom adjacent to the carbon radical is known to stabilize the radical through hyperconjugation and electron delocalization. Therefore, we used 4-thia-L-lysine as the inhibitor, in which the C4 of lysine is replaced by a sulfur atom adjacent to the carbon radical at the C5 position. This inhibitor is known for Porphyromonas gingivalis LAM (PgLAM), with previous EPR studies showing that it promotes Co(III)-C bond homolysis and induces conformational changes.23-25\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUpon anaerobic addition of 4-thialysine to holo TtLAM, the EPR spectrum revealed the formation of a transient intermediate that persisted for up to 30 seconds (Fig. 3). This intermediate lasted longer than was observed in PgLAM,36 possibly due to the increased thermostability of the TtLAM. Low-spin, square-planar Co(II) compounds, including derivatives of coenzyme B12, typically exhibit g┴ values between 2.2-3.0 and A|| around 100 G.37-39 However, the EPR spectrum of the transient species captured showed g┴ = 2.12 and A|| = 45 G, accompanied by a distinctive 8-line hyperfine pattern corresponding to the nuclear spin (I = 7/2) of the Co atom, indicating the presence of a spin-coupled Co(II)/radical pair, a feature commonly observed in AdoCbl-dependent enzymes.40 The radical could be located either at the PLP-lysine conjugate (as a Sub\u0026bull; radical), or associated with the adenosyl group after Co(III)-C homolysis (as a Ado\u0026bull; radical) (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo pinpoint the radical\u0026apos;s location, 13C-labeled ATP was reacted with hydroxocobalamin to synthesize 13C-AdoCbl following a previously reported procedure.41 The resulting 13C-AdoCbl was then incorporated into TtLAM along with PLP to reconstitute the holoenzyme. The reaction of 13C-AdoCbl LAM with 4-thialysine under the same conditions was freeze-quenched at 30 seconds, and the EPR spectrum revealed no significant hyperfine changes in the 13C-labeled samples compared to those with natural isotope abundance samples (Fig. 3). This absence of hyperfine changes confirmed that the radical is located at the PLP-substrate adduct rather than at the C5\u0026apos; position of adenosine. In PgLAM, 13C-labeled 4-thia-L-lysine was used to probe the radical location, and the radical captured was located at the PLP-substrate adduct.36 Our results aligned with the reported observations in a different LAM and excluded the possibility of Ado\u0026bull; radical as a co-existing reactive species in the transient intermediate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Co(II)/ Sub\u0026bull; radical pair interacts through dipole-dipole and exchange interactions, both of which are influenced by orbital overlap and spin polarization effects.40 The dipole-dipole interaction is particularly valuable for structural analysis because it follows a 1/r\u0026sup3; dependence on the distance between the Co(II) and Sub\u0026bull; radical pair. For distances greater than 4 \u0026Aring;, this interaction breaks the degeneracy of the spin states in the absence of an external magnetic field, resulting in zero-field splitting, which is described by axial D and rhombic E terms. The D term is distance-dependent and is described by the equation D = D₀/r\u0026sup3;, where r (\u0026Aring;) is the inter-spin distance, and D₀ = 2.785 \u0026times; 10⁴ G\u0026Aring;\u0026sup3;.40 By determining the D term, the distance between the Co(II) and Sub\u0026bull; radical pair can be calculated. The EPR spectrum of Co(II)/ Sub\u0026bull; radical pair was simulated as an axial low-spin Co(II) species (g┴Co = 2.27, g||Co = 2.0, A┴Co = 20 G, A||Co = 100 G),37 strongly coupled with an isotropic carbon radical species (giso,C = 2.003), with zero-field splitting parameters D = -152.4 G (Fig. 3). This corresponded to a calculated distance of 5.7 \u0026Aring; between the Co(II) and Sub\u0026bull; radical.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further explore how distance affects the EPR spectra, we simulated the spectra by varying the distance between the Co(II) and Sub\u0026bull; radical (Fig. S7). For distances less than 7 \u0026Aring;, no significant changes were observed in the simulated EPR spectra. This is consistent with the observed g┴ values of 2.12 for the Co(II)/ Sub\u0026bull; radical pair, which is smaller than the typical g┴ values of 2.2-3.0 for low-spin, square-planar Co(II) compounds. The \u0026nbsp;decrease in g┴ was simulated to result from exchange interactions between the two spin systems at distances below 7 \u0026Aring;, leading to g-value averaging and eventually converging to a triplet state.40 Distances longer than 7 \u0026Aring; led to significant changes in the simulated EPR spectra due to the weakening of both dipole-dipole and exchange interactions as the spatial separation increases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2, the AdoCbl and PLP cofactors are separated by a distance of approximately 25 \u0026Aring;. The EPR calculated distance of less than 7 \u0026Aring; between the Co(II) and Sub\u0026bull; radical suggested a conformational change upon substrate binding, which brings the AdoCbl cofactor and the PLP-Sub adduct closer. Therefore, spectroscopic evidence indicated that the \u0026lsquo;Closed\u0026rsquo; state in LAM involves not only a TIM barrel conformational change similar to MCM,14 but also a more substantial movement that reduces spatial distance between the two cofactors from approximately 25 \u0026Aring; to less than 7 \u0026Aring;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCryo-EM Structure of Holo TtLAM \u0026nbsp;in \u0026lsquo;Close\u0026rsquo; State. With the timing of the transient intermediate identified by EPR spectroscopy, we captured the \u0026lsquo;Closed\u0026rsquo; state of TtLAM by freeze-quenching the mixture of holo TtLAM and 4-thia-L-lysine in liquid ethane at the 30 s timepoint. The cryo-EM structure of TtLAM in the \u0026lsquo;Closed\u0026rsquo; state was solved at 3.0 \u0026Aring; resolution (Fig. 4). In one \u0026beta; subunit (\u0026beta;\u0026apos;), the dimerization subdomain is well-resolved, while the Rossman domain is invisible, possibly because the mixture was quenched during the dynamic motion of the subunit during catalysis. In the other \u0026beta; subunit, both the dimerization and Rossman domains were fully resolved (Fig. 4a). Superimposed structures of TtLAM in the \u0026lsquo;Open\u0026rsquo; and \u0026lsquo;Closed\u0026rsquo; conformations revealed a substantial 64.1\u0026deg; rotation of the Rossman domain, while the dimerization domains and \u0026alpha; subunits remained static (Fig. 4c). Since the AdoCbl cofactor is anchored to the Rossman domain, this rotation shifted the cofactor by an unprecedented 25.5 \u0026Aring;, bringing it closer to the PLP-substrate adduct (Fig. 4b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe active site of TtLAM in the \u0026lsquo;Closed\u0026rsquo; state is shown in Fig. 5. The cobalamin remains bound to the Rossmann domain in a \u0026ldquo;base-off/His-on\u0026rdquo; conformation, with His133\u0026beta; serving as the lower axial ligand to the cobalt center (Fig. 5a). The position of the PLP cofactor is unchanged between the \u0026lsquo;Open\u0026rsquo; and \u0026lsquo;Closed\u0026rsquo; states. In both conformations, PLP is clamped between Tyr237\u0026alpha; and Tyr264\u0026alpha; through \u0026pi;-\u0026pi; stacking interactions between the pyridine ring of PLP and the tyrosine rings on either side. The phosphate group of PLP is locked by electrostatic interactions with Arg185\u0026alpha; and Arg269\u0026alpha;. The key difference in the PLP cofactor between the \u0026lsquo;Open\u0026rsquo; and \u0026lsquo;Closed\u0026rsquo; forms lies in its interaction with Lys144\u0026beta;. In the \u0026lsquo;Open\u0026rsquo; state, PLP is covalently bound to Lys144\u0026beta; via an imine bond (Fig. 2b). However, in the \u0026lsquo;Closed\u0026rsquo; state, Lys144\u0026beta; is replaced by 4-thia-L-lysine through a transimination reaction. Therefore, in \u0026lsquo;Closed\u0026rsquo; state, there is no covalent linkage between PLP and the Rossmann domain. This substitution frees Lys144\u0026beta;, allowing rotation of the Rossmann domain independent from PLP. Notably, the distance between Co(II) and the C5 position of 4-thia-L-lysine is 6.3 \u0026Aring; (Fig. 5a), consistent with the distance of \u0026lt; 7 \u0026Aring; between Co(II) and the Sub\u0026bull; radical as measured by EPR spectroscopy (Fig. 3). Overall, our cryo-EM structure provides direct structural information to confirm the long-existing hypothesis of Rossmann domain rotation in LAM activation.\u003c/p\u003e\n\u003cp\u003eAnother key question in the LAM mechanism is how the enzyme controls the highly reactive Ado\u0026bull; radical formed upon Co(III)\u0026ndash;C bond homolysis. The bond dissociation energy (BDE) for the adenosine C5\u0026prime;\u0026ndash;H bond is estimated to be between 94 and 101 kcal/mol.42 The highly reactive carbon-centered Ado\u0026bull; radical could potentially react with various C-H bonds or other functional groups within the enzyme\u0026apos;s substrate binding site, leading to side reactions that might compromise the integrity of the catalytic site. The cryo-EM structure of the \u0026lsquo;Closed\u0026rsquo; state revealed that the cleaved adenosine is tightly restricted in the active site through hydrogen bonds with Lys371\u0026alpha; and Asp299\u0026alpha; (Fig. 5b), preventing rotation around the C\u0026ndash;N single bond between adenine and ribose and consequently any conformation or position change of the ribose group. Additionally, with the PLP locked in place, the substrate lysine is positioned between the cobalamin and adenosine. The C5\u0026apos; of adenosine is precisely aligned with the C5 and C6 positions of 4-thia-lysine, maintaining distances of 4.7 \u0026Aring; and 5.1 \u0026Aring;, respectively (Fig. 5c). This controlled spatial arrangement ensures that the Ado\u0026bull; radical remains properly oriented toward the substrate, preventing unwanted side reactions and protecting the catalytic site. Moreover, the alignment ensures the correct shift of the amino group between C5 and C6 to produce the desired product, illustrating how TtLAM controls the selectivity of the isomerization reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSystematic Mutation of the Hydrogen Bond Network Around Adenosine. In addition to the precise positioning of adenosine by Asp299\u0026alpha;, Glu412\u0026alpha;, and Lys371\u0026alpha; in the \u0026lsquo;Closed\u0026rsquo; form (Fig. 5), the hydrogen bond network may also play an integral role in facilitating the Co(III)-C homolysis. To test this hypothesis, we systematically mutated the residues involved in the hydrogen bond network and monitored Co(III)-C bond homolysis using EPR spectroscopy. In the E412Q mutant, the carboxylic side chain of Glu was replaced with the amide in Gln, which could weaken H-bond or alter local electric field and consequently affect both the adenosine and the periphery of the corrin ring. The mutant was loaded with PLP and AdoCbl and reacted with 4-thia-L-lysine. The reaction mixture was quenched at 30 s, and the corresponding ERP spectra was silent, indicating that the subtle change induced by the mutation prevented the homolysis of the Co(III)-C bond and the formation of the Co(II)/ Sub\u0026bull; radical pair (Fig. 3). Similarly, the reaction mixture of the K371Q mutant was also EPR silent (Fig. 3). The reaction mixture of D299N mutant, which was designed similar to E412Q, produced radical pair EPR signals similar to the WT but with much lower intensity (Fig. 3), indicating that Co(III)-homolysis occurred but with reduced yield. These mutation studies confirmed the critical role of the hydrogen bond network and local electric field in the active site in facilitating the Co(III)-C bond cleavage.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSubstrate binding can trigger conformational changes in proteins, enabling or enhancing their catalytic activity. This mechanism is widely employed by nature in many enzymatic processes and is fundamental to a range of biological functions. Most enzymes undergo only minute conformational changes, typically involving small shifts in the geometry of catalytic residues, usually less than 1 \u0026Aring;.\u003csup\u003e43\u003c/sup\u003e In some enzymes, more significant movements of the binding residues occur, especially when these residues are located on the surface loops.\u003csup\u003e44\u003c/sup\u003e Domain alternation refers to larger-scale conformational changes, where different domains of an enzyme shift between distinct positions or orientations.\u003csup\u003e45\u003c/sup\u003e Although rare in metalloproteins, domain alternation also can play a crucial role in regulating their catalytic activity. For example, in methionine synthase (MetH), movement of the cobalamin-binding cap domain creates a new interface between cobalamin and the S-adenosylmethionine (SAM)-binding domain, facilitating the transfer of a methyl group from SAM to cobalamin.\u003csup\u003e46,47\u003c/sup\u003e Similarly, in ribonucleotide reductase (RNR), substrate binding to the \u0026alpha; subunit induces domain rearrangements that bring the \u0026alpha; and \u0026beta; subunits into close proximity, enabling the 32 \u0026Aring;-long radical transfer pathway essential for RNR activity.\u003csup\u003e48\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur findings showcased domain alternation as a mechanism for activating the metallocofactor in \u003cem\u003eTt\u003c/em\u003eLAM, as supported by cryo-EM structures and ERP spectroscopy. Simply binding the cofactor AdoCbl to the protein scaffold does not activate the Co(III)-C bond.\u003csup\u003e11\u003c/sup\u003e To cleave the Co(III)-C bond of 30 kcal dissociation energy and achieve the enzymatic turnover rate, the activation process must reduce the energy required for bond cleavage by 17 kcal/mol. As demonstrated by x-ray and our cryo-EM structures, in the \u0026lsquo;Open\u0026rsquo; state, PLP is located in the TIM barrel domain (\u0026alpha; subunit) and is also covalently linked with Lys144\u0026beta; of the Rossmann domain within the \u0026beta; subunit. (Fig. 2b). Therefore, PLP acts as an anchor, connecting the TIM barrel and Rossmann domains and enforcing an unconventional orientation of the Rossmann domain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cryo-EM structure of the \u0026lsquo;Closed\u0026rsquo; state revealed that, upon substrate binding, transimination frees Lys144\u0026beta;, dismantling the PLP-mediated anchoring between the Rossmann and TIM barrel domains. This release triggers a substantial conformational shift, including a 64.1-degree rotation of the Rossmann domain, which moves the AdoCbl cofactor by 25.5 \u0026Aring; (Fig. 4c) into a new binding site. In the new site, the dissociated adenosine is stabilized by a hydrogen bond network involving Asp299\u0026alpha;, Glu412\u0026alpha;, and Lys371\u0026alpha; from the TIM barrel. (Fig. 5). These hydrogen bonds, all located on one side of adenosine opposite the cobalamin, not only restrict the adenosine\u0026rsquo;s position after dissociation, but may also exert a \u0026quot;pull\u0026quot; effect on the adenosyl group even before the Co(III)-C bond cleavage, shifting it away from the cobalamin and weakening the Co(III)-C bond, thereby decreasing the bond dissociation energy and promoting its homolytic cleavage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis substrate-controlled metallocofactor activation mirrors mechanisms observed in heme and non-heme iron proteins. For instance, in cytochrome P450 enzymes, substrate binding forces the heme iron to transition from a six-coordinate to a five-coordinate state, resulting in a spin-state switch from low-spin to high-spin. The high-spin state facilitates electron transfer to the Fe\u0026sup3;⁺ center to form the Fe\u0026sup2;⁺ reduced state, which is necessary for oxygen activation and formation of the highly reactive Fe(IV)=O species.\u003csup\u003e26\u003c/sup\u003e Similarly, in \u0026alpha;-ketoglutarate (\u0026alpha;-KG)-dependent non-heme iron dioxygenases, the binding of \u0026alpha;-KG and the substrate induces a shift from a six-coordinate to a five-coordinate iron center, creating a vacant site essential for oxygen binding and activation.\u003csup\u003e27\u003c/sup\u003e A similar concept applies here: without the substrate, the Co(III)-C bond will not be activated to generate the highly reactive Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical. Co(III)-C bond activation only occurs in the presence of the substrate, ensuring that the generated Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical can be quenched by the substrate C-H bond. However, this process is distinct in that the activation is driven by protein domain alternation and metallocofactor relocation rather than changes in the coordination of the metallocofactors.\u003c/p\u003e\n\u003cp\u003eRadical-based chemistry offers exceptional reactivity but carries the inherent risk of side reactions due to the highly reactive nature of radical intermediates, which can deactivate the enzyme. This work illustrated the elegant natural design of LAM that controls radical generation through substrate binding and mitigates radical damage via binding site design. The sophisticated hydrogen-bonding network surrounding adenosine after Co(III)-C bond homolysis effectively directs the radical toward the substrate. These structures elegantly demonstrate how the protein fold harnesses radical-based chemistry, emphasizing both the precision and protective strategies employed by the enzyme.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur investigation of \u003cem\u003eTt\u003c/em\u003eLAM using cryo-EM and EPR provided crucial insights into the enzyme\u0026rsquo;s activation mechanism. The cryo-EM structures experimentally demonstrated, for the first time, a 64.1-degree rotation of the Rossmann domain induced by substrate binding, which relocates the AdoCbl cofactor by 25.5 \u0026Aring;. This movement is essential for activating the Co(III)-C bond to form the highly reactive Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical. In the \u0026lsquo;Closed\u0026rsquo; state, key hydrogen-bond network involving Lys371\u0026alpha;, Asp299\u0026alpha;, and Glu412\u0026alpha; around adenosine ensure the precise orientation of the Ado\u003csup\u003e\u0026bull;\u003c/sup\u003e radical toward the PLP-conjugated lysine, controlling HAA at the C5 or C6 positions of the substrate and preventing harmful side reactions. By employing 4-thialysine, the transient Sub\u003csup\u003e\u0026bull;\u003c/sup\u003e was freeze quenched, and EPR spectra confirmed the proximity of the cobalt center and the radical located on the substrate. This work highlighted the sophisticated structural strategies employed by LAM to efficiently and safely harness radical-based chemistry, offering broader implications for understanding metallocofactor activation in other radical-based enzymes.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the U.S. National Institute of General Medical Sciences (R35GM155016 to S.T.) for funding the research described in this work. We also thank Dr. Tatsuo Kurihara (Kyoto University) for generously providing the plasmid for \u003cem\u003eTt\u003c/em\u003eLAM expression and Dr. Jorge Escalante (University of Georgia) for the plasmid for \u003cem\u003eCobA\u003c/em\u003e expression. Additionally, we are grateful to Purdue University\u0026apos;s Research Instrumentation Center for access to EPR, facilitated by the Amy Instrumentation Facility, Department of Chemistry, under the supervision of Dr. Michael Everly and Dr. Aloke Bera. We also extend our thanks to the Purdue Cryo-EM Facility for providing access to the Titan Krios instrument.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBanerjee, R. Radical Carbon Skeleton Rearrangements:\u0026thinsp; Catalysis by Coenzyme B12-Dependent Mutases. \u003cem\u003eChem. Rev.\u003c/em\u003e\u003cstrong\u003e103\u003c/strong\u003e, 2083-2094, (2003).\u003c/li\u003e\n\u003cli\u003eBrown, K. L. Chemistry and Enzymology of Vitamin B12. \u003cem\u003eChem. Rev.\u003c/em\u003e\u003cstrong\u003e105\u003c/strong\u003e, 2075-2150, (2005).\u003c/li\u003e\n\u003cli\u003eDowling, D. P., Croft, A. K. \u0026amp; Drennan, C. L. 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Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex. \u003cem\u003eScience\u003c/em\u003e\u003cstrong\u003e368\u003c/strong\u003e, 424-427, (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-5313240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5313240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lysine 5,6-aminomutase (LAM) is a radical-based enzyme that catalyzes the reversible migration of an amino group between the C5 and C6 positions of lysine. This reaction is mediated by the coenzymes adenosylcobalamin (AdoCbl) and pyridoxal 5’-phosphate (PLP). Our study investigated the activation mechanism of AdoCbl in LAM. Using cryo-electron microscopy (cryo-EM), we resolved structures of LAM in both its ‘Open’ and ‘Closed’ states, revealing a substantial 64.1-degree rotation of the Rossmann domain upon substrate binding, which shifts the AdoCbl cofactor 25.5 Å towards PLP. This large conformational shift enables Co(III)-C homolysis and adenosine radical (Ado•) formation. Following domain alternation, adenosine is stabilized by a key hydrogen bond network composed of Lys371α, Asp299α, and Glu412α, while PLP is anchored by hydrogen bonds and electrostatic interactions with two Tyr and two Arg residues, securing the substrate lysine in place. The enzyme pocket is precisely designed to ensure the proper orientation and positioning of Ado• and PLP-substrate complex, facilitating efficient hydrogen abstraction from the substrate after the formation of the highly reactive Ado• radical, which is crucial for maintaining catalytic efficiency. This study provided the first structural information supporting the long-existing hypothesis of Rossmann domain rotation as part of the LAM activation process, shedding light on the enzymes' conformational dynamics and illustrating how nature employs domain movements to activate metallocofactors and control radical chemistry.","manuscriptTitle":"Catalysis in Motion: Large-Scale Domain Alternation Enables Co(III)-C Bond Homolysis in Lysine 5,6-Aminomutase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 06:44:40","doi":"10.21203/rs.3.rs-5313240/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":"def19d65-38b1-4585-b83b-6d09709e383f","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39316896,"name":"Biological sciences/Biochemistry/Bioinorganic chemistry/Metalloproteins"},{"id":39316897,"name":"Biological sciences/Biochemistry/Enzyme mechanisms"}],"tags":[],"updatedAt":"2024-11-27T18:55:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-05 06:44:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5313240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5313240","identity":"rs-5313240","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}