Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine

Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine | 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 Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine Feng Long, Wei Huang, Jun Song, Tianxue Sun, Yue He, Zixin Deng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3357770/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Selenium is a multi-functional trace element essential for diverse organisms. SenB is a Se -glycosyltransferase that incorporates selenium into small molecules in the selenoneine biosynthesis pathway and is also the only known Se -glycosyltransferase in nature. Although the biochemical function of SenB has been investigated, its substrate specificity, structure, and catalytic mechanism remain unclear. Here, we revealed that SenB exhibits sugar donor specificity and promiscuity and can utilize six UDP-sugars to generate selenosugars. The crystal structures of SenB complexed with four different UDP-sugars were solved. The residues N20, T23, and E231 were proven as the key elements that determine the sugar donor promiscuity of SenB. Structure-guided mutagenesis further revealed a novel catalytic triad H58/D86/K158 in SenB, which accounts for the C-Se glycosidic bond formation and Se-P bond cleavage during the Se -glycosylation process. Furthermore, we mined, functionally and structurally characterized two other novel Se -glycosyltransferase, Cb SenB and Rs SenB, which also exhibit sugar donor promiscuity. Biological sciences/Chemical biology/Enzyme mechanisms Biological sciences/Structural biology/X-ray crystallography Biological sciences/Chemical biology/Enzymes Selenium Se-glycosyltransferase Crystal structure Se-glycosylation Catalytic mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Selenium (Se) was firstly discovered from sulfur ore in 1817 1 . Se has been shown as an essential trace element for maintaining normal life activities in humans and animals, as well as being involved in a variety of physiological processes within the living body 2 – 4 . Se exhibits broad pharmacological activities, including anti-inflammatory 5 , anti-tumor 6 , antioxidant 7 , and hepatoprotective effects 8 . Se deficiency can cause the development of a variety of diseases, such as cardiovascular disease 9 , liver disease 10 , thyroid disease 11 , and diabetes 12 . Selenium enters the metabolic pathway of the human body by forming selenoproteins and selenonucleic acid biopolymers 2 . Selenoproteins carry Se in the form of selenocysteine, and the selenocysteine synthetase SelA utilizes selenophosphate (SeP) with the assistance of the cofactor pyridoxal-5-phosphate (PLP) to convert seryl-tRNA Sec to selenocysteinyl-tRNA Sec 13 , 14 . Selenonucleic acids carry Se in the form of 5-methylaminomethyl-2-selenouridine, and rhodanese enzyme SelU utilizes SeP to convert 2-thiouridine to 2-selenouridine 15 . Selenoneine (SEN), a Se analog of ergothioneine, was initially identified in the blood of tuna and exhibits greater antioxidant effect than ergothioneine 16 – 18 . Recently, it was reported that Se is incorporated into small molecules through a specific SEN biosynthetic pathway in Variovorax paradoxus DSM 30034 19 , which is recognized as the third way adopted by Se for its incorporation into organic matter in nature 2 , 19 . The SEN biosynthesis gene cluster encodes three proteins SenA, SenB, and SenC, among which SenB catalyzes the generation of the key intermediate selenoglucose product using SeP synthesized by SenC 19 (Fig. 1 ). SenB was identified as the first reported selenosugar synthase, and the third reported enzyme catalytically generating the C-Se bond in addition to SelA and SelU 13 – 15 , 19 . The catalytic function of SenB was proven distinct from that of SelA and SelU, in the way that SenB is capable of utilizing UDP-Glc, UDP-GlcNAc, or UDP-GalNAc to catalyze the generation of the corresponding selenosugars in the absence of cofactors 13 , 19 . The reaction catalyzed by SenB is more similar to the catalytic process of glycosyltransferases, because of which SenB is also considered a Se -glycosyltransferase (SeGT). Glycosyltransferases are a class of enzymes that transfer sugar groups from sugar donors to sugar acceptors. Glycosyltransferases are widespread in living organisms in abundance, and are involved in the process of glycosylation modification of primary and secondary metabolites 20 – 22 . Glycosyltransferases can be further categorized by their various three-dimensional structures (GT-A, GT-B, GT-C and GT-D), or by different glycosidic bonds formed in the products ( O -/ N -/ S -/C-glycosyltransferases) 20,23 . SenB is the only Se -glycosyltransferase reported so far. In contrast to other types of glycosyltransferases, SenB possesses dual function in a two-step catalytic reaction, catalyzing both the formation of the C-Se glycosidic bond and the subsequent cleavage of the Se-P bond to generate the final product 19 , 20 . Since the catalytic function and sequence similarity of SenB are fairly low compared with the other reported enzymes, its structure and catalytic mechanism remain uncertain and demand in-depth investigation. In this work, we performed structure-function analysis as well as the catalytic mechanism deduction of SenB using functional characterization, crystallization, and structure-based mutagenesis. Moreover, we mined and identified two other novel SeGTs, Cb SenB and Rs SenB, functional and structural findings of which greatly supported our proposed catalytic mechanisms of sugar donor promiscuity and Se -glycosylation of SeGTs. Results Probing sugar donor selectivity of SenB Previous studies showed that SenB can utilize UDP-GlcNAc and UDP-GalNAc as sugar donors to generate corresponding selenosugars 19 . UDP-GlcNAc and UDP-GalNAc are a class of sugar donors with large spatial dimensions due to the presence of an N -acetyl group on the C2′ of the sugar moiety. Therefore, we hypothesized that SenB may also utilize other types of sugar donors. We selected ten different sugar donors, including UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-Rha, UDP-Xyl, UDP-GlcA, UDP-GalA, ADP-Glc, and GDP-Man, and utilized SenC-produced SeP as a sugar acceptor to test the sugar donor specificity of SenB (Fig. 2 a, 2 b and Extended Data Fig. 1 ). The monobromobimane (mBBr) derivatives with a single Se atom were yielded from the final product of the enzymatic reaction using the thiol-labeling reagent and detected by HPLC-DAD/MS. The results showed that SenB has the promiscuity of sugar donors (Fig. 2 d and Extended Data Fig. 2 ). In addition to the three sugar donors UDP-Glc, UDP-GlcNAc and UDP-GalNAc reported in the literature, SenB can also utilize three other UDP-sugars, including UDP-Gal, UDP-Rha and UDP-Xyl. SenB also has specificity of sugar donor, and can only utilize the UDP form of sugar donors, but not the ADP or GDP form of sugar donors such as ADP-Glc and GDP-Man. Lower activity of SenB was shown towards UDP-Rha and UDP-Xyl compared to UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. The dissociation constants ( K d ) of SenB against different sugar donors varied in a range from nM to µM (Fig. 2 c and Extended Data Fig. 3 ). It reveals that SenB prefers UDP-GlcNAc > UDP-GalNAc > UDP-Glc > UDP-Gal > UDP-Rha > UDP-Xyl. Given the chemical structure and catalytic activity of the sugar donor, it suggests that the N -acetyl group of C2′ and the hydroxymethyl group of C5′ on the sugar moiety account for the catalytic efficiency of SenB. Selenosugars, which have a variety of promising biological activities, are mainly obtained by chemical syntheses that face disadvantageous limitations such as complex reaction conditions, poor yields and selectivity, and lack of diversity 24 – 26 . The sugar donor promiscuity of SenB could alleviate the drawbacks of chemically synthesized selenosugar, and SenB likely serves as a promising and potentially applicable enzyme for the efficient and green synthesis of selenosugars with different structures. Overall crystal structure of SenB To elucidate the structural basis of the substrate recognition and catalytic mechanism of SenB, we solved the structures of SenB complexed with various sugar donors, including the ternary complex SenB/UDP-Glc/PO 4 3− (1.95 Å), as well as three binary complexes SenB/UDP-GlcNAc (1.88 Å), SenB/UDP-GalNAc (1.64 Å) and SenB/UDP-Rha (2.35Å) (Fig. 3 a and Extended Data Fig. 4 and Supplementary Table 1 ). In all solved crystal structures, each asymmetric unit contains three copies of SenB molecules, which are unlikely functionally related due to lack of the protein-protein interfaces between them. It is in agreement with the size-exclusion chromatography result that SenB is monomeric in solution. The structure of monomeric SenB consists of two domains containing Rossmann-like fold, the N-terminal domain (NTD; residues 1-130, 311–331) and the C-terminal domain (CTD; residues146-310), which are connected by a loop (residues 131–145). The NTD contains 6 parallel β-folds and 9 α-helices, whereas the CTD contains 5 parallel β-folds and 8 α-helices. The active site of SenB is present in a narrow cleft formed by the face-to-face apposition of the NTD and CTD, with UDP-sugar bound to the CTD and PO 4 3− bound to the NTD (Fig. 3 b ) . These structural features are similar to those of the GT-B type of glycosyltransferases 20 , and thus SenB is likely a GT-B glycosyltransferase. According to the structural similarity analysis using the DALI server 27 , the top two hits most similar to SenB were, a sucrose synthase (PDB ID: 6KIH) from Thermosynechococcus vestitus 28 with an RMSD (root mean square deviation) of 2.4 Å for Ca atoms and 18% sequence similarity, and a GT-B glycosyltransferases BshA (PDB ID: 6N1X) from Staphylococcus aureus 29 with an RMSD of 2.9 Å for Ca atoms and 12% sequence similarity, respectively. It suggests that the catalytic mechanism of SenB may differ significantly from that of the reported enzymes. Structural mechanisms for sugar donor binding and promiscuity of SenB Limited structural information on sugar-enzyme complexes leading to mechanism dissection of the sugar donor promiscuity has been a challenge for glycosyltransferase studies 35 – 37 . In this work, the electron densities of the four different sugar moieties of the UDP-sugars were well defined with great success in our solved complex structures of SenB (Fig. 4 a- 4 d). Based on the crystal structure of SenB/UDP-Glc/PO 4 3− , we further constructed the complexed models of SenB/UDP-Gal and SenB/UDP-Xyl (Fig. 4 e, 4 f). Structural superposition of SenB/UDP-Glc, SenB/UDP-GlcNAc, SenB/UDP-GalNAc and SenB/UDP-Rha indicated that the spatial positions of the UDP moieties of the four sugar donors overlapped well ( Extended Data Fig. 5 a, 5 b). The interaction between SenB and UDP was examined in details ( Extended Data Fig. 5 c and Supplementary Table 2 ). Different from most of the GT-B glycosyltransferases with known structures that mainly used π-π interactions to stabilize uracil 30 – 34 , SenB held its bound uracil by forming numerous hydrogen bonds alternatively. A substantial space limitation around uracil was observed in SenB, leading to difficult accommodation within SenB of the nitrogenous bases with a large molecular backbone such as adenosine (A) and guanosine (G). It could structurally explain that SenB only utilized the UDP-sugars, but not the ADP-sugars or the GDP-sugars. Alanine mutagenesis screening showed that mutations of the amino acids that directly interact with UDP affected the catalytic activity of SenB. For example, the single alanine mutants K158A and E239A showed a nearly complete loss of catalytic activity. The catalytic activity of both the mutant L209A and T214A with reduced spatial hindrance was increased by about 1.5-fold ( Extended Data Fig. 5 d). To reveal the structural mechanism of the sugar donor promiscuity of SenB, we performed an in-depth comparative analysis of the interaction between the sugar moiety and SenB (Fig. 4 g). It was proposed earlier that the chemical groups on the C2' and C5' positions of the sugar moiety play crucial roles for the catalytic efficiency of SenB. We next compared the interactions involving the C2′ position of the sugar moiety. It showed that E231 of SenB forms tight interactions with the C2′ groups of the sugar in all six complex structures. Therefore, it was speculated that E231 might also be important for the catalytic activity of SenB. The mutagenesis results showed that the relative catalytic activities of the mutant E231A for different UDP-sugars were significantly reduced (< 20%) ( Fig. 4 h ) . Despite the hydrophobic interaction between V157 and the C-2 position of UDP-GalNAc or UDP-GlcNAc, SenB maintained a certain catalytic activity after V157 was mutated to different types of amino acids (A/F/I/M/R/K) ( Fig. 4 h ) . Comparative analysis revealed that N20 and T23 were essential in stabilizing the C5′ hydroxymethyl group of sugar moiety. Although only moderate decreases in the relative catalytic activity were observed in the mutants N20A or T23A, the catalytic activities of the double mutant N20A/T23A were reduced to less than 10% in the presence of different sugar donors ( Fig. 4 h ) . It suggested that N20 and T23 are also catalytically important residues of SenB as to different sugar donors. Further combinatorial mutagenesis showed that the triad mutant N20A/T23A/E231A resulted in a complete loss of the relative catalytic activity of SenB for all the tested sugar donors ( Fig. 4 h ) . These results suggested that the core site composed of N20/T23/E231 determines the sugar donor promiscuity of SenB. Residues interacting with other sites of the sugar moiety do not affect the sugar donor promiscuity of SenB, although they could affect the catalytic activity of SenB to some extent. For instance, the catalytic activities were still partially preserved but slightly varied with different sugar donors in the single and double mutants of Q131 and H235, the residues of which interact with C4' of the sugar moiety ( Fig. 4 h ) . Structural basis for SeP binding and catalytic mechanism of SenB To investigate the catalytic mechanism of SenB, it is necessary to clarify the structural basis of SeP binding. Based on the structure of SenB/UDP-Glc/PO 4 3− , we constructed a structural model of the ternary complex SenB/UDP-Glc/SeP ( Extended Data Fig. 6 a, 6 d). It was shown that SeP was bound in a narrow pocket near UDP-Glc, consisting of a series of hydrophilic residues (N20, H58, R61, T83, T85, R155), which stabilize SeP in the pocket by forming abundant hydrogen bonds ( Extended Data Fig. 6 b, 6 c). Single alanine mutations of the above hydrophilic residues only hampered the activities of SenB to some extent. However, when we performed multipoint mutations to further disrupt the pocket hydrophilicity, the mutants N20A/T85A, T83A/T85A and N20A/T83A/T85A resulted in a complete loss of activity ( Extended Data Fig. 5 e). These results suggested that the hydrophilicity of the sugar acceptor binding pocket ensures the stable binding of SeP and is essential for the catalytic activity of SenB. Formation of the Se-C glycosidic bond is the first step towards producing selenosugars by SeGTs. SenB could generate 1-seleno-β-D-glucose from UDP-α-D-glucose 19 , suggesting that the configuration at anomeric carbon of the sugar moiety is flipped during the catalytic process. Thus, the generation of Se-C glycosidic bonds by SenB likely adopts an S N 2-like mechanism 20 . Deprotonation of the receptor is initially important during the process of forming glycosidic bonds following an S N 2-like mechanism 20 . A putative catalytic dyad His58-Asp86, critical residues highly conserved in GT-B type glycosyltransferases that assist in the deprotonation of substrates 20 , 38 – 40 ( Extended Data Fig. 7 ), was identified near the SeP binding site of SenB (Fig. 5 a). Thus, the catalytic dyad His58-Asp86 in SenB may assist formation of the Se-C glycosidic bond. In an alternative way, the deprotonation of SeP could be possibly achieved spontaneously by the selenol group (-SeH) of SeP performing nucleophilic attack in its selenolate form, because both SeP and in particular -SeH are highly electronegative at neutral pH (the p K a values are 4.19 for SeP and 0.98 for -SeH) 41 (Fig. 5 b). Mutagenesis showed reduced but not complete loss of enzymatic activity in the mutants H58A, H58Q, and H58D, and almost complete loss of activity in the mutants H58F and D86A (Fig. 5 c). These results further supported that SeP may undergo both spontaneous and non-spontaneous deprotonation in SenB, and formation of the C-Se glycosidic bond, first step of the reaction catalyzed by SenB, may follow an S N 2-like mechanism (Fig. 5 f). Like SelA and SelU, the catalytic reaction of SenB involves the cleavage of the Se-P bond, but the specific process remains unclear. SelA cleaves the Se-P bond via K258 in the SeP binding pocket with the participation of water molecules 42 . Coincidentally, a residue K158 was found near the SeP binding pocket of SenB (Fig. 5 a). The catalytic activity of the K158A mutant was almost completely lost, indicating that K158 was crucial for SenB to function ( Extended Data Fig. 5 d). In the structure of SenB/UDP-Glc/PO 4 3− , a molecule of water is indeed stabilized between K158 and PO 4 3− , leading to a water bridge formed between K158 and PO 4 3− (Fig. 5 a). Therefore, K158 in SenB likely performs an equivalent function as K258 in SelA. The amino side chain of K158 captures the proton from the water molecule, making it a negatively charged nucleophilic agent to attack the positively charged P atoms of Se-P, which leads to the cleavage of the Se-P bond to produce the final product of selenosugar (Fig. 5 f). The hypothesized role of K158 during the Se-P bond cleavage was proven by examining the catalyzed products of the functionally impaired SenB mutant K158A (Fig. 5 d). Using UDP-Glc, UDP-GlcNAc and UDP-GalNAc as sugar donors, the corresponding key selenophosphosugar intermediates were all detected by LC-MS for catalysis by the K158A mutant (Fig. 5 e). To further consolidate our hypothesis, mutation of K158 to different types of amino acids were introduced and most of the mutants resulted in a complete loss of catalytic activity of SenB. Only the mutants K158R, K158H, and K158N with alkaline side chains or with amino side chains retained weak catalytic activity (Fig. 5 b). These results further indicated that K158 is indeed involved in the Se-P cleavage and plays an irreplaceable role in the catalytic reaction of SenB. This catalytic mechanism of SenB by catalyzing formation of the C-Se glycosidic bond and cleavage of the Se-P bond in use of the catalytic triad H58/D86/K158 is completely different from that of the other glycosyltransferases that have been reported so far. Mining and characterization of SenB-like enzymes We did gene mining for the homologous SeGTs in the NCBI database using SenB as a probe, and more than 200 SenB-like genes were found (> 55% similarity), which were mainly distributed in β-proteobacteria. Sequence conservation analysis of these SenB-like enzymes revealed that the putative catalytic triad H58/D86/K158 exhibit a high degree of conservation, which further supports our hypothesis on the catalytic mechanism of SenB ( Extended Data Fig. 8 ). Besides, there is a highly conserved “EGGAHV” motif found related to sugar donor binding in SenB-like enzymes (Fig. 4 g and Extended Data Fig. 7 ). One of the three key amino acids that determines the sugar donor promiscuity of SenB, E231, is located within this conserved motif. The other two key amino acids, N20/T23, are also highly conserved among these SenB-like enzymes (Extended Data Fig. 7) . Therefore, we suspect that these SenB-like enzymes may all have a certain degree of sugar donor promiscuity. We selected two SenB-like enzymes, Cb SenB (GenBank: RYF17368.1; 65.5% sequence similarity) derived from Comamonadaceae bacterium and RsSenB (GenBank: MBC7468551.1, 65.2% sequence similarity) derived from Ramlibacter sp. , for further functional characterization (Extended Data Fig. 1 c- 1 f and Supplementary Table 3) . Sequence alignment showed that Cb SenB and Rs SenB contain the putative catalytic triad H58/D86/K158 as well as the key EGGAHV motif (Fig. 6 a). Catalytic activity analysis showed that Cb SenB and Rs SenB recognize sugar donors similarly to SenB, and that both utilize UDP-Glc, UDP-GlcNAc, UDP-Gal, and UDP-GalNAc to generate the corresponding selenosugar (Fig. 6 b). The catalytic activities of these three enzymes for UDP-Glc, UDP-GlcNAc, and UDP-Gal were similar, but Rs SenB utilized UDP-GalNAc with a stronger capacity than SenB and Cb SenB. To further confirm our hypothesis on the catalytic mechanism of Se -glycosylation, we solved the crystal structure of Rs SenB (PDB ID: 8K5U) in apo form at a resolution of 2.15 Å ( Supplementary Table 4 ). Unlike SenB, the asymmetric unit of the crystal contains two Rs SenB molecules (Fig. 6 c). The monomer structure of Rs SenB is highly similar to that of SenB, with an RMSD of 0.99 Å for Ca atoms (Fig. 6 d). The spatial positions of the two residues D86/K158 are highly overlapped compared the structures of SenB and Rs SenB. Despite the spatial position shift of residue H58, both are still located in the center of catalytic activity (Fig. 6 e). In addition, the mutagenesis results of the catalytic triad in Rs SenB and Cb SenB were consistent with those of SenB (Fig. 6 f). These results not only provided two new SeGTs, but also further demonstrated the universal effects of the key amino acids N20/T23/E231 on the promiscuity of sugar donor and the catalytic triad H58/D86/K158 on Se -glycosylation of SenB. Discussion SenB is the only functional identified SeGT in nature, but its structure and catalytic mechanism are completely unknown. In this work, substrate specificity investigations showed that SenB has sugar donor promiscuity and specificity to utilize six sugar donors in the form of UDP. Structures of SenB in complex with four sugar donors were solved including SenB/UDP-Glc/PO 4 3− , SenB/UDP-GlcNAc, SenB/UDP-GalNAc and UDP-Rha. Structural analysis reveals that loss of π-π interactions, leading to increased steric hindrance in the neighborhood of uracil, is the major factor in the specific recognition of UDP-form sugar donors by SenB. Structural comparison and mutagenesis revealed the critical amino acids N20, T23, and E231 that control sugar donor promiscuity of SenB. Furthermore, catalytic mechanism investigations identified the catalytic triad H58/D86/K158 in SenB that are involved in the formation of the C-Se bond and the cleavage of Se-P, and the catalytic process of Se -glycosylation was deduced. Finally, two new SeGTs, Rs SenB and Cb SenB were mined, and functionally and structurally characterized. These results revealed the structure of SenB, and the possible mechanism of Se -glycosylation, providing insights into the diversity of C-Se bond formation as well as Se-P bond cleavage in nature, and theoretical guidance for structure-based engineering modification of SeGT for selenosugars or drugs synthesis. Declarations ACKNOWLEDGMENT We thank the staffs from BL19U1 beamlines of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for their assistance during data collection, and Prof B.Y from Yangzhou University for providing selenoglucose as a control. This work was supported by grant to F.L. from the National Key Research and Development Program of China (2021YFA0909500), grant to W.H. from National Natural Science Foundation of China (Grant No. 82304333 to W.H.). and grant to F.L. from the Fundamental Research Funds for the Central Universities (2042019kf0185). Author Contributions W.H. and F.L. designed the experiments. 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Biol. 3 , 657–662 (2007). Alexander, J. A. N., & Locher, K. P. Emerging structural insights into C-type glycosyltransferases. Curr. Opin. Struc. Biol. 79 , 102547 (2023). Yang, Y., Liang, Y., Cui, F. et al. UDP-Glycosyltransferases in Edible Fungi: Function, Structure, and Catalytic Mechanism. Fermentation 9 , 164 (2023). Yao, J., Xing. X., Yu, L., et al. Structure-function relationships in plant UDP-glycosyltransferases. Ind. Crop. Prod. 189 , 115784 (2022). Yang, Q. et al. Holistic Prediction of p K a in Diverse Solvents Based on Machine Learning Approach. Angew. Chem. Int. Ed. 59 , 19282-19291 (2020). Itoh, Y., Bröcker, M. J., Sekine, S. I., Hammond, G., Suetsugu, S., Söll, D. & Yokoyama, S. Decameric SelA•tRNASec Ring Structure Reveals Mechanism of Bacterial Selenocysteine Formation. Science 340 , 75-78 (2013). Methods Materials and reagents All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. mBBr, UDP-Glc, UDP-GlcNAc, UDP-Gal, UDP-GalNAc, UDP-Xyl, UDP-Rha, UDP-GlcA, GDP-Man and ADP-Glc were purchased from Meryer Biotechnology (Shanghai, China). Cloning Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme Biotechnology (Nanjing, China). Hieff Clone Plus Multi One Step Cloning Kit was purchased from Yeasen Biotechnology (Shanghai, China). Restriction Enzymes was purchased from New England BioLabs (Ipswich, MA, USA). Codon-optimized gene fragments were purchased from Tsingke (Beijing, China). All crystallization materials and reagents were purchased from Hampton Research (Laguna Niguel, CA, USA). Acetonitrile and formic acid of HPLC grade were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Plasmid construction for protein expression All protein coding DNA sequences were codon-optimized for expression in E. coli , and commercially synthesized. The DNA fragments were then amplified using Super-Fidelity DNA Polymerase, and ligated with the Nde I and Hin dIII linearized pET28a vector using one step cloning kit following the manufacturer’s instructions. Ligation mixtures were transformed into chemically competent E. coli TOP10 by heat shock and plated onto LB agar containing 50 μg/mL kanamycin. Single colony was picked and cultured for plasmid extraction. The constructions confirmed by Sanger sequencing were later used for protein expression. Site-Directed Mutagenesis Site-directed mutagenesis of SenB was performed using the polymerase chain reaction (PCR) with primers designed to generate the desired mutations ( Supplementary Table5 ). The wild-type pET28a-SenB plasmid was used as the PCR template. PCR was set up with a Phanta Max Super-Fidelity DNA Polymerase in a Biorad C1000 Thermal Cycler. After digestion of the template DNA with Dpn I for 2 h in 37°C, the PCR products were transformed into the E. coli TOP10 competent cell. All of the mutations were confirmed by the Sanger sequencing. Expression and purification of SenB, SenC, Rs SenB, Cb SenB and mutants All proteins were individually produced in E. coli Rosetta (DE3) cells. Cells transformed with corresponding expression plasmids were cultured in 1 L of LB medium (1% Tryptone, 0.5% Yeast extract, 1% NaCl) containing 50 µg/mL kanamycin, shaken at 220 rpm and 37°C until the OD 600 reached 0.6, then induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and growth at 18°C for 18 h. Cells were pelleted by centrifugation (SORVALL LYNX 4000, Thermo Scientific) at 6,000 rpm for 10 min, resuspended in 80 mL of lysis buffer consisting of 20 mM Tris-HCl, 300 mM NaCl, pH 7.5, and disrupted by a high-pressure homogenizer (EmulsiFlex-C3, AVESTIN, Canada) at 12,000 psi. The lysate was clarified by centrifugation at 15,000g for 45 min, and the supernatant was loaded onto a gravity column pre-equilibrated in lysis buffer with 5 mL Ni-NTA affinity resins (GenScript, Nanjing, China). The column was washed sequentially with lysis buffer containing 20 mM imidazole for 10 CV (column volume), 50 mM imidazole for 6 CV, and then the target proteins were eluted with lysis buffer containing 300 mM imidazole for 3 CV. The eluted proteins were further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column (GE Healthcare) in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM dithiothreitol (DTT). The peak fractions containing target protein were collected and examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the purified protein was concentrated to 7.5 mg/mL using an Amicon Ultra-30 K filter (Millipore), flash-frozen in the liquid nitrogen, and stored at − 80°C for later use. Enzyme activity assay of SenB, RsSenB, CbSenB and mutants Under anaerobic conditions, the reactions were performed in a final volume of 50 µL, containing 20 µM SenC, 20 µM SenB, Rs SenB, Cb SenB or mutants, 2 mM DTT, 2 mM ATP, 1 mM Na 2 Se, and 2 mM of UDP-sugar. All of materials were prepared in buffer consisting of 50 mM Tri-HCl, 20 mM KCl and 5 mM MgCl 2 , pH 7.2. Experimental reactions were prepared in an identical manner, except that different mutants of SenB and UDP-sugar were used. After a 6 h incubation period at room temperature, reactions were removed from the glovebox and exposed to atmosphere for 30 min to oxidize any unreacted Na 2 Se. The reactions were then quenched with 50 µL of 10 mM ice cold mBBr in MeCN, followed by incubation in dark at room temperature for an additional 30 min to allow the completeness of derivatization with mBBr. Finally, the supernatants were collected after centrifugation at 12,000 rpm for 30 min, and analyzed by HPLC-UV/DAD and HPLC-MS. HPLC and LC − MS analysis The HPLC analysis was performed on a Shimadzu-LC-20AT (Japan) with an Ultimate® XB-C18 column (4.6 mm×250 mm I.D., 5 µm, Welch Materials, Inc., China) at a flow rate of 0.8 mL/min, using the mobile phase of (A) 0.1% formic acid in deionized H 2 O and (B) 100% MeCN. The gradient settings for separating the products and substrates were 0 − 20 min 10% B to 50% B, 20 − 25 min 50% B to 100% B, 25 − 28 min 100% B to 10% B, and 28 − 35 min 10% B to 10% B. The products were further confirmed using an LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) The MS/MS analysis was carried out in a positive ionization mode with 35% relative collision energy. The relative activities of the mutants were determined by HPLC and calculated by the product's peak area dividing the wild-type's peak area. All experiments were performed in triplicate. Microscale thermophoresis assay The purified SenB protein was exchanged into test buffer consisting of 25 mM Hepes, 150 mM NaCl, 0.05% Tween 20, 1 mM DTT, pH 7.5 for the microscale thermophoresis (MST) experiments. The protein was diluted to a final concentration of 1 µM in test buffer, then a 100 nM MO-L018 RED-tris-NTA dye solution was added to protein solution. The protein and dye mixture were mixed well and incubated at 4°C in the dark for 30 min. The labeled protein was obtained by centrifugation at 12,000 rpm for 10 min. The binding affinities between substrates and proteins were analyzed on a Monolith NT.115 instrument (Nanotemper Technologies). Different concentrations of UDP-sugars were serially diluted from the stocks using test buffer (the premade stocks were 40 µM UDP-Glc, 50 µM UDP-Gal, 10 µM UDP-GlcNAc, 20 µM UDP-GalNAc, 1 mM UDP-Rha, and 10 mM UDP-Xyl, respectively). The equal volumes of labeled protein were added to various concentrations of UDP-sugar solutions in a final volume of 10 µL. After being incubated at 4°C in the dark for 30 min, the reaction mixtures were loaded into standard treated capillaries (Monolith NT.115 series capillaries MO-K022) and analyzed by MST at medium MST power and auto-detect excitation power with a laser-on time of 2.5 s and a detection temperature of 25°C. The K d values were calculated using MO. Affinity Analysis Software from three independent thermophoresis measurements. Selenophosphosugar intermediate analysis Under anaerobic conditions, enzyme activity assays of the K158A mutant of SenB were performed in an identical manner, except being supplied with different UDP-sugars (UDP-Glc/UDP-GlcNAc/UDP-GalNAc). After a 6 h incubation period at room temperature, reactions were removed from the glovebox and exposed to atmosphere for 30 min to oxidize any unreacted Na 2 Se. The reactions were then quenched with 50 µL MeCN, and centrifuged at 12,000 rpm for 30 min. The supernatants were analyzed by LC-MS. The LC was performed with an COSMOSIL® PBr column (4.6 mm×250 mm I.D.) at a flow rate of 0.3 mL/min, using the mobile phase of (A) 10 mM NH 4 Ac in deionized H 2 O and (B) 100% MeCN. The Elution settings were 0 − 25 min 5% B. The products were further confirmed using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc.) Protein crystallization The purified SenB protein (7.5 mg/mL) was incubated with UDP-Glc (5mM), UDP-GlcNAc (5 mM), UDP-GalNAc (5 mM), UDP-Rha (5 mM) for 30 min on ice, and the purified Rs SenB protein (7.5 mg/mL) was incubated with UDP-GalNAc (5mM), before set-up of the crystallization trays. Crystals of SenB/UDP-Glc/PO 4 3− were observed at 18 ℃ within 3–4 d using the hanging drop vapor diffusion method by mixing 0.8 µL of protein with 0.8 µL of the reservoir solution (0.2 M Ammonium sulfate, 0.1 M Hepes (pH 7.5), and 20% (w/v) polyethylene glycol 8000, 10% (v/v) 2-Propanol). The crystals of SenB/UDP-GalNAc were obtained in the reservoir solution containing 0.1 M Bis-Tris (pH 6.5), and 20% (w/v) polyethylene glycol 5000-MME. The crystals of SenB/UDP-GlcNAc and SenB/UDP-Rha were obtained in the reservoir solution containing 0.1 M Hepes (pH 7.0), and 15% (w/v) polyethylene glycol 20000. The crystals of Rs SenB/UDP-GalNAc were obtained in the reservoir solution containing 0.2 M Ammonium citrate dibasic, and 20% (w/v) polyethylene glycol 3350. All crystals were harvested in the same reservoir solution supplemented with 20% (w/v) glycerol as the cryo-protectant and flash-frozen in the liquid nitrogen. Data collection and structure determination The crystallographic data sets were collected on the beamlines 19U1 at the Shanghai Synchrotron Radiation Facility (SSRF) 43 . The diffraction images were processed using XDS 44 . The structures of SenB and Rs SenB were solved by molecular replacement using a Phaser from the CCP4 suite 45 , and the alphafold2 46 predicted structure of SenB was used as the searching model. The models of the SenB complexes were built initially using AutoBuild 47 and manually using Coot 48 . The iterative refinement and structure validation were done using Phenix 49 . Structure analysis Structural visualization analysis and figure preparation were made with Protein-ligand interaction profiler 50 and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). Sequence alignments were created using Clustal Omega 51 , ESPript 52 , and WebLogo 53 . Molecular docking Autodock 4.0 54 was used to build the structures of SenB/UDP-Xyl, SenB/UDP-Gal, and SenB/UDP-Glc/SeP, using SenB/UDP-Glc/PO 4 3− as the template. References 43 Zhang, W.-Z. et al. The protein complex crystallography beamline (BL19U1) at the Shanghai Synchrotron Radiation Facility. Nucl. Sci. Tech. 30 , 170 (2019). 44 Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66 , 125–132 (2010). 45 Bunkoczi, G. et al. Phaser.MRage: automated molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 69 , 2276–2286 (2013). 46 Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583–589 (2021). 47 Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64 , 61–69 (2008). 48 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66 , 486–501 (2010). 49 Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66 , 213–221 (2010). 50 Adasme, M. F. et al. PLIP 2021: expanding the scope of the protein-ligand interaction profiler to DNA and RNA. Nucleic Acids Res. 49 , W530-W534 (2021). 51 Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47 , W636-W641 (2019). 52 Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42 , W320-324 (2014). 53 Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res . 14 , 1188–1190 (2004). 54 Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31 , 455–461 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files SI20230915F.docx Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine ExtendedDataFigs.docx Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2024 Read the published version in Nature Communications → 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. 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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-3357770","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":236252283,"identity":"47ae95e0-2993-46ac-ac83-811dc80631ea","order_by":0,"name":"Feng Long","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACPhBRwWDDYABi8BCjhQ1EnGFII13LYVK0sPcefnGg4nzidokExgdv2xjkzQlq4TmXZnHgzO3EnTMSmA3ntjEY7mwgpEUix8z4Y9vt3A03EtikedsYEgwOEKHF4OC/cyAt7L+J1WL84GDDAbAtzMRp4TljxnDgWHL9hjMPmyXnnJMw3EBICz97j/GHAzV2xgbHkw9+eFNmI0/QFrDbIDRjA5CQIKweCJg/EKVsFIyCUTAKRi4AAGIKQiLI10BnAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Long","suffix":""},{"id":236252284,"identity":"7e1ac3b9-b690-437c-bc40-c7f098d401dc","order_by":1,"name":"Wei Huang","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Huang","suffix":""},{"id":236252285,"identity":"890548a4-9ec5-455c-aaeb-94cc79aba508","order_by":2,"name":"Jun Song","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Song","suffix":""},{"id":236252286,"identity":"fc8f769c-9664-4427-ae1a-74d3d198e3cb","order_by":3,"name":"Tianxue Sun","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianxue","middleName":"","lastName":"Sun","suffix":""},{"id":236252287,"identity":"1c5d3620-c28b-433b-afc0-3bb1b01fef7f","order_by":4,"name":"Yue He","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"He","suffix":""},{"id":236252288,"identity":"6b2890b9-1f57-48fa-92bf-f247be8571f3","order_by":5,"name":"Zixin Deng","email":"","orcid":"https://orcid.org/0000-0003-0724-3390","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zixin","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2023-09-15 08:01:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3357770/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3357770/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-46065-6","type":"published","date":"2024-02-23T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":43842610,"identity":"6d0b11e0-0f14-464a-878d-6ade8748f020","added_by":"auto","created_at":"2023-09-28 15:41:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosynthetic pathway for SEN\u003c/strong\u003e. SenB catalyzed generation of the key intermediate selenoglucose product using SeP synthesized by SenC.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/f3b439ef474066abaf4ce13b.png"},{"id":43842613,"identity":"8e0351a3-f9a2-4847-aa75-17209dba1374","added_by":"auto","created_at":"2023-09-28 15:41:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProbing sugar donor selectivity of SenB\u003c/strong\u003e. \u003cstrong\u003ea,\u003c/strong\u003e \u003cstrong\u003ei\u003c/strong\u003e) SenB catalyzes the synthesis of selenosugar using SeP; \u003cstrong\u003eii\u003c/strong\u003e) Derivatization of selenosugar using mBBr. \u003cstrong\u003eb, \u003c/strong\u003eChemical structures of sugar donors.\u003cstrong\u003e c, \u003c/strong\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values of SenB for binding of different sugar donors. \u003cstrong\u003ed, \u003c/strong\u003eHPLC-UV/DAD analysis of the mBBr derivatives of the SenB products from the reactions using different sugar donors at 391 nm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/896b6c9df907827f9c0dd618.png"},{"id":43844282,"identity":"9a57e24e-0dc0-426b-891c-b0d747eea56e","added_by":"auto","created_at":"2023-09-28 15:57:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":538797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall structure of SenB. a,\u003c/strong\u003e Cartoon diagram of the three copies of the ternary SenB complex formed by SenB with UDP-Glc and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e within one asymmetric unit. \u003cstrong\u003eb,\u003c/strong\u003e Cartoon diagram of the monomer of the ternary SenB complex formed by SenB with UDP-Glc and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e. The N-terminal domain of SenB is marked with a green box and the C-terminal domain with a blue box. The secondary α-helix and β-shift structures are labeled in black font. UDP-Glc as well as PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3- \u003c/sup\u003eare indicated by sticks.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/1ec8eeaeb36efa95c4cae4c1.png"},{"id":43842616,"identity":"3c79b3d9-3df1-4ddc-a831-3d42341c42df","added_by":"auto","created_at":"2023-09-28 15:41:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural mechanisms for sugar donor binding and promiscuity of SenB.\u003c/strong\u003e \u003cstrong\u003ea-f, \u003c/strong\u003eInteraction of the sugar moieties of different sugar donors with SenB. The 2\u003cem\u003eFo-Fc\u003c/em\u003e electron density maps of Glc/GlcNAc/Rha/GalNAc were contoured at 2.0 σ (\u003cstrong\u003ea-d\u003c/strong\u003e). Hydrogen bonds are indicated by black dashed lines and hydrophobic interactions are indicated by magenta dashed lines.\u003cstrong\u003e g, \u003c/strong\u003eresidues that interact with different sites of the sugar donor.\u003cstrong\u003e h, \u003c/strong\u003eRelative catalytic activities of the SenB mutants for different sugar donors.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/a3e93cf4ad6cd79cd194b4b0.png"},{"id":43843461,"identity":"750739fd-b7fd-4628-ba8b-5ce311fc9043","added_by":"auto","created_at":"2023-09-28 15:49:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":291488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatalytic mechanism of SenB.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Catalytic activity centers and potential catalytic triads of SenB. Hydrogen bonds are represented by black dashed lines, water bridges by green dashed lines, and water molecules by red spheres.\u003cstrong\u003e b, \u003c/strong\u003eThe p\u003cem\u003eK\u003c/em\u003ea value of SeP and -SeH. \u003cstrong\u003ec,\u003c/strong\u003e Relative catalytic activity of the SenB mutants using UDP-Glc as the sugar donor.\u003cstrong\u003e d, \u003c/strong\u003eThe K158A mutant halts the reaction of the Se-P bond cleavage.\u003cstrong\u003e e,\u003c/strong\u003e LC-MS detection of the selenophosphosugar intermediates from catalysis by the K158A mutant. \u003cstrong\u003ef, \u003c/strong\u003eProposed reaction mechanism for \u003cem\u003eSe\u003c/em\u003e-glycosylation of SenB.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/21e85890da4f827d4129ac7b.png"},{"id":43842612,"identity":"bff397ae-2a5a-4f6e-a1d2-379a51205247","added_by":"auto","created_at":"2023-09-28 15:41:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":546724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of SenB-like enzymes. a,\u003c/strong\u003e Protein sequence comparison of \u003cem\u003eCb\u003c/em\u003eSenB, \u003cem\u003eRs\u003c/em\u003eSenB and SenB. N20/T23, H58/D86/K158 and the EGGAHV motif are labeled with black triangle.\u003cstrong\u003e b, \u003c/strong\u003eHPLC-UV/DAD analysis of the catalytic activity of \u003cem\u003eCb\u003c/em\u003eSenB and\u003cem\u003e Rs\u003c/em\u003eSenB. \u003cstrong\u003ec, \u003c/strong\u003eOverall structure of \u003cem\u003eRs\u003c/em\u003eSenB within one asymmetric unit. \u003cstrong\u003ed, \u003c/strong\u003eStructural superposition of the structures of \u003cem\u003eRs\u003c/em\u003eSenB (magenta) and SenB/UDP-GlcNAc (cyan). \u003cstrong\u003ee, \u003c/strong\u003eSpatial positions of H58/D86/K158 in SenB (cyan) and \u003cem\u003eRs\u003c/em\u003eSenB (magenta). \u003cstrong\u003ef, \u003c/strong\u003eHPLC-UV/DAD analysis of the catalytic activity of catalytic triad mutants in\u003cem\u003e Rs\u003c/em\u003eSenB and\u003cem\u003e Cb\u003c/em\u003eSenB.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/3be1d09f307383815579e37f.png"},{"id":51579540,"identity":"f0522de8-47f2-4fb1-9a0b-966b756bb5ad","added_by":"auto","created_at":"2024-02-24 08:08:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3280980,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/9899420c-1a72-4926-a9b8-b13bc427d293.pdf"},{"id":43843462,"identity":"50da787f-a9d0-4c5d-9108-3b7c3bdcae6a","added_by":"auto","created_at":"2023-09-28 15:49:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40665,"visible":true,"origin":"","legend":"\u003cp\u003eStructural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine\u003c/p\u003e","description":"","filename":"SI20230915F.docx","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/33319bc4136ff6c3a9ece79a.docx"},{"id":43842617,"identity":"3f405ebd-b4b5-4871-a68c-27b924290028","added_by":"auto","created_at":"2023-09-28 15:41:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4390660,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigs.docx","url":"https://assets-eu.researchsquare.com/files/rs-3357770/v1/d6af5c0f56a901fcfeb3f774.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSelenium (Se) was firstly discovered from sulfur ore in 1817\u003csup\u003e1\u003c/sup\u003e. Se has been shown as an essential trace element for maintaining normal life activities in humans and animals, as well as being involved in a variety of physiological processes within the living body\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Se exhibits broad pharmacological activities, including anti-inflammatory\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, anti-tumor\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, antioxidant\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and hepatoprotective effects\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Se deficiency can cause the development of a variety of diseases, such as cardiovascular disease\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, liver disease\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, thyroid disease\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and diabetes\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Selenium enters the metabolic pathway of the human body by forming selenoproteins and selenonucleic acid biopolymers\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Selenoproteins carry Se in the form of selenocysteine, and the selenocysteine synthetase SelA utilizes selenophosphate (SeP) with the assistance of the cofactor pyridoxal-5-phosphate (PLP) to convert seryl-tRNA\u003csub\u003eSec\u003c/sub\u003e to selenocysteinyl-tRNA\u003csub\u003eSec\u003c/sub\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Selenonucleic acids carry Se in the form of 5-methylaminomethyl-2-selenouridine, and rhodanese enzyme SelU utilizes SeP to convert 2-thiouridine to 2-selenouridine\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSelenoneine (SEN), a Se analog of ergothioneine, was initially identified in the blood of tuna and exhibits greater antioxidant effect than ergothioneine\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Recently, it was reported that Se is incorporated into small molecules through a specific SEN biosynthetic pathway in \u003cem\u003eVariovorax paradoxus\u003c/em\u003e DSM 30034 \u003csup\u003e19\u003c/sup\u003e, which is recognized as the third way adopted by Se for its incorporation into organic matter in nature\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The SEN biosynthesis gene cluster encodes three proteins SenA, SenB, and SenC, among which SenB catalyzes the generation of the key intermediate selenoglucose product using SeP synthesized by SenC\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). SenB was identified as the first reported selenosugar synthase, and the third reported enzyme catalytically generating the C-Se bond in addition to SelA and SelU\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The catalytic function of SenB was proven distinct from that of SelA and SelU, in the way that SenB is capable of utilizing UDP-Glc, UDP-GlcNAc, or UDP-GalNAc to catalyze the generation of the corresponding selenosugars in the absence of cofactors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The reaction catalyzed by SenB is more similar to the catalytic process of glycosyltransferases, because of which SenB is also considered a \u003cem\u003eSe\u003c/em\u003e-glycosyltransferase (SeGT).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGlycosyltransferases are a class of enzymes that transfer sugar groups from sugar donors to sugar acceptors. Glycosyltransferases are widespread in living organisms in abundance, and are involved in the process of glycosylation modification of primary and secondary metabolites\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Glycosyltransferases can be further categorized by their various three-dimensional structures (GT-A, GT-B, GT-C and GT-D), or by different glycosidic bonds formed in the products (\u003cem\u003eO\u003c/em\u003e-/\u003cem\u003eN\u003c/em\u003e-/\u003cem\u003eS\u003c/em\u003e-/C-glycosyltransferases)\u003csup\u003e20,23\u003c/sup\u003e. SenB is the only \u003cem\u003eSe\u003c/em\u003e-glycosyltransferase reported so far. In contrast to other types of glycosyltransferases, SenB possesses dual function in a two-step catalytic reaction, catalyzing both the formation of the C-Se glycosidic bond and the subsequent cleavage of the Se-P bond to generate the final product\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Since the catalytic function and sequence similarity of SenB are fairly low compared with the other reported enzymes, its structure and catalytic mechanism remain uncertain and demand in-depth investigation.\u003c/p\u003e \u003cp\u003eIn this work, we performed structure-function analysis as well as the catalytic mechanism deduction of SenB using functional characterization, crystallization, and structure-based mutagenesis. Moreover, we mined and identified two other novel SeGTs, \u003cem\u003eCb\u003c/em\u003eSenB and \u003cem\u003eRs\u003c/em\u003eSenB, functional and structural findings of which greatly supported our proposed catalytic mechanisms of sugar donor promiscuity and \u003cem\u003eSe\u003c/em\u003e-glycosylation of SeGTs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eProbing sugar donor selectivity of SenB\u003c/h2\u003e\n\u003cp\u003ePrevious studies showed that SenB can utilize UDP-GlcNAc and UDP-GalNAc as sugar donors to generate corresponding selenosugars\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. UDP-GlcNAc and UDP-GalNAc are a class of sugar donors with large spatial dimensions due to the presence of an \u003cem\u003eN\u003c/em\u003e-acetyl group on the C2\u0026prime; of the sugar moiety. Therefore, we hypothesized that SenB may also utilize other types of sugar donors. We selected ten different sugar donors, including UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-Rha, UDP-Xyl, UDP-GlcA, UDP-GalA, ADP-Glc, and GDP-Man, and utilized SenC-produced SeP as a sugar acceptor to test the sugar donor specificity of SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb \u003cstrong\u003eand Extended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The monobromobimane (mBBr) derivatives with a single Se atom were yielded from the final product of the enzymatic reaction using the thiol-labeling reagent and detected by HPLC-DAD/MS. The results showed that SenB has the promiscuity of sugar donors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed \u003cstrong\u003eand Extended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition to the three sugar donors UDP-Glc, UDP-GlcNAc and UDP-GalNAc reported in the literature, SenB can also utilize three other UDP-sugars, including UDP-Gal, UDP-Rha and UDP-Xyl. SenB also has specificity of sugar donor, and can only utilize the UDP form of sugar donors, but not the ADP or GDP form of sugar donors such as ADP-Glc and GDP-Man. Lower activity of SenB was shown towards UDP-Rha and UDP-Xyl compared to UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. The dissociation constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) of SenB against different sugar donors varied in a range from nM to \u0026micro;M (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec \u003cstrong\u003eand Extended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). It reveals that SenB prefers UDP-GlcNAc\u0026thinsp;\u0026gt;\u0026thinsp;UDP-GalNAc\u0026thinsp;\u0026gt;\u0026thinsp;UDP-Glc\u0026thinsp;\u0026gt;\u0026thinsp;UDP-Gal\u0026thinsp;\u0026gt;\u0026thinsp;UDP-Rha\u0026thinsp;\u0026gt;\u0026thinsp;UDP-Xyl. Given the chemical structure and catalytic activity of the sugar donor, it suggests that the \u003cem\u003eN\u003c/em\u003e-acetyl group of C2\u0026prime; and the hydroxymethyl group of C5\u0026prime; on the sugar moiety account for the catalytic efficiency of SenB. Selenosugars, which have a variety of promising biological activities, are mainly obtained by chemical syntheses that face disadvantageous limitations such as complex reaction conditions, poor yields and selectivity, and lack of diversity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The sugar donor promiscuity of SenB could alleviate the drawbacks of chemically synthesized selenosugar, and SenB likely serves as a promising and potentially applicable enzyme for the efficient and green synthesis of selenosugars with different structures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eOverall crystal structure of SenB\u003c/h2\u003e\n\u003cp\u003eTo elucidate the structural basis of the substrate recognition and catalytic mechanism of SenB, we solved the structures of SenB complexed with various sugar donors, including the ternary complex SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e (1.95 \u0026Aring;), as well as three binary complexes SenB/UDP-GlcNAc (1.88 \u0026Aring;), SenB/UDP-GalNAc (1.64 \u0026Aring;) and SenB/UDP-Rha (2.35\u0026Aring;) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea \u003cstrong\u003eand Extended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003eand Supplementary Table\u0026nbsp;1\u003c/strong\u003e). In all solved crystal structures, each asymmetric unit contains three copies of SenB molecules, which are unlikely functionally related due to lack of the protein-protein interfaces between them. It is in agreement with the size-exclusion chromatography result that SenB is monomeric in solution. The structure of monomeric SenB consists of two domains containing Rossmann-like fold, the N-terminal domain (NTD; residues 1-130, 311\u0026ndash;331) and the C-terminal domain (CTD; residues146-310), which are connected by a loop (residues 131\u0026ndash;145). The NTD contains 6 parallel \u0026beta;-folds and 9 \u0026alpha;-helices, whereas the CTD contains 5 parallel \u0026beta;-folds and 8 \u0026alpha;-helices. The active site of SenB is present in a narrow cleft formed by the face-to-face apposition of the NTD and CTD, with UDP-sugar bound to the CTD and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e bound to the NTD (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e. These structural features are similar to those of the GT-B type of glycosyltransferases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and thus SenB is likely a GT-B glycosyltransferase. According to the structural similarity analysis using the DALI server\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, the top two hits most similar to SenB were, a sucrose synthase (PDB ID: 6KIH) from \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e with an RMSD (root mean square deviation) of 2.4 \u0026Aring; for Ca atoms and 18% sequence similarity, and a GT-B glycosyltransferases BshA (PDB ID: 6N1X) from \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e with an RMSD of 2.9 \u0026Aring; for Ca atoms and 12% sequence similarity, respectively. It suggests that the catalytic mechanism of SenB may differ significantly from that of the reported enzymes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eStructural mechanisms for sugar donor binding and promiscuity of SenB\u003c/h2\u003e\n\u003cp\u003eLimited structural information on sugar-enzyme complexes leading to mechanism dissection of the sugar donor promiscuity has been a challenge for glycosyltransferase studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In this work, the electron densities of the four different sugar moieties of the UDP-sugars were well defined with great success in our solved complex structures of SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Based on the crystal structure of SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, we further constructed the complexed models of SenB/UDP-Gal and SenB/UDP-Xyl (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). Structural superposition of SenB/UDP-Glc, SenB/UDP-GlcNAc, SenB/UDP-GalNAc and SenB/UDP-Rha indicated that the spatial positions of the UDP moieties of the four sugar donors overlapped well (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). The interaction between SenB and UDP was examined in details (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec \u003cstrong\u003eand Supplementary Table\u0026nbsp;2\u003c/strong\u003e). Different from most of the GT-B glycosyltransferases with known structures that mainly used \u0026pi;-\u0026pi; interactions to stabilize uracil\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, SenB held its bound uracil by forming numerous hydrogen bonds alternatively. A substantial space limitation around uracil was observed in SenB, leading to difficult accommodation within SenB of the nitrogenous bases with a large molecular backbone such as adenosine (A) and guanosine (G). It could structurally explain that SenB only utilized the UDP-sugars, but not the ADP-sugars or the GDP-sugars. Alanine mutagenesis screening showed that mutations of the amino acids that directly interact with UDP affected the catalytic activity of SenB. For example, the single alanine mutants K158A and E239A showed a nearly complete loss of catalytic activity. The catalytic activity of both the mutant L209A and T214A with reduced spatial hindrance was increased by about 1.5-fold (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eTo reveal the structural mechanism of the sugar donor promiscuity of SenB, we performed an in-depth comparative analysis of the interaction between the sugar moiety and SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg). It was proposed earlier that the chemical groups on the C2' and C5' positions of the sugar moiety play crucial roles for the catalytic efficiency of SenB. We next compared the interactions involving the C2\u0026prime; position of the sugar moiety. It showed that E231 of SenB forms tight interactions with the C2\u0026prime; groups of the sugar in all six complex structures. Therefore, it was speculated that E231 might also be important for the catalytic activity of SenB. The mutagenesis results showed that the relative catalytic activities of the mutant E231A for different UDP-sugars were significantly reduced (\u0026lt;\u0026thinsp;20%) \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e. Despite the hydrophobic interaction between V157 and the C-2 position of UDP-GalNAc or UDP-GlcNAc, SenB maintained a certain catalytic activity after V157 was mutated to different types of amino acids (A/F/I/M/R/K) \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e. Comparative analysis revealed that N20 and T23 were essential in stabilizing the C5\u0026prime; hydroxymethyl group of sugar moiety. Although only moderate decreases in the relative catalytic activity were observed in the mutants N20A or T23A, the catalytic activities of the double mutant N20A/T23A were reduced to less than 10% in the presence of different sugar donors \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e. It suggested that N20 and T23 are also catalytically important residues of SenB as to different sugar donors. Further combinatorial mutagenesis showed that the triad mutant N20A/T23A/E231A resulted in a complete loss of the relative catalytic activity of SenB for all the tested sugar donors \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e. These results suggested that the core site composed of N20/T23/E231 determines the sugar donor promiscuity of SenB. Residues interacting with other sites of the sugar moiety do not affect the sugar donor promiscuity of SenB, although they could affect the catalytic activity of SenB to some extent. For instance, the catalytic activities were still partially preserved but slightly varied with different sugar donors in the single and double mutants of Q131 and H235, the residues of which interact with C4' of the sugar moiety \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eStructural basis for SeP binding and catalytic mechanism of SenB\u003c/h2\u003e\n\u003cp\u003eTo investigate the catalytic mechanism of SenB, it is necessary to clarify the structural basis of SeP binding. Based on the structure of SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, we constructed a structural model of the ternary complex SenB/UDP-Glc/SeP (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). It was shown that SeP was bound in a narrow pocket near UDP-Glc, consisting of a series of hydrophilic residues (N20, H58, R61, T83, T85, R155), which stabilize SeP in the pocket by forming abundant hydrogen bonds (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). Single alanine mutations of the above hydrophilic residues only hampered the activities of SenB to some extent. However, when we performed multipoint mutations to further disrupt the pocket hydrophilicity, the mutants N20A/T85A, T83A/T85A and N20A/T83A/T85A resulted in a complete loss of activity (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). These results suggested that the hydrophilicity of the sugar acceptor binding pocket ensures the stable binding of SeP and is essential for the catalytic activity of SenB.\u003c/p\u003e\n\u003cp\u003eFormation of the Se-C glycosidic bond is the first step towards producing selenosugars by SeGTs. SenB could generate 1-seleno-\u0026beta;-D-glucose from UDP-\u0026alpha;-D-glucose\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, suggesting that the configuration at anomeric carbon of the sugar moiety is flipped during the catalytic process. Thus, the generation of Se-C glycosidic bonds by SenB likely adopts an S\u003csub\u003eN\u003c/sub\u003e2-like mechanism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Deprotonation of the receptor is initially important during the process of forming glycosidic bonds following an S\u003csub\u003eN\u003c/sub\u003e2-like mechanism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. A putative catalytic dyad His58-Asp86, critical residues highly conserved in GT-B type glycosyltransferases that assist in the deprotonation of substrates \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eExtended Data Fig.\u0026nbsp;7\u003c/strong\u003e), was identified near the SeP binding site of SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Thus, the catalytic dyad His58-Asp86 in SenB may assist formation of the Se-C glycosidic bond. In an alternative way, the deprotonation of SeP could be possibly achieved spontaneously by the selenol group (-SeH) of SeP performing nucleophilic attack in its selenolate form, because both SeP and in particular -SeH are highly electronegative at neutral pH (the p\u003cem\u003eK\u003c/em\u003ea values are 4.19 for SeP and 0.98 for -SeH) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Mutagenesis showed reduced but not complete loss of enzymatic activity in the mutants H58A, H58Q, and H58D, and almost complete loss of activity in the mutants H58F and D86A (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). These results further supported that SeP may undergo both spontaneous and non-spontaneous deprotonation in SenB, and formation of the C-Se glycosidic bond, first step of the reaction catalyzed by SenB, may follow an S\u003csub\u003eN\u003c/sub\u003e2-like mechanism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003eLike SelA and SelU, the catalytic reaction of SenB involves the cleavage of the Se-P bond, but the specific process remains unclear. SelA cleaves the Se-P bond via K258 in the SeP binding pocket with the participation of water molecules\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Coincidentally, a residue K158 was found near the SeP binding pocket of SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The catalytic activity of the K158A mutant was almost completely lost, indicating that K158 was crucial for SenB to function (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). In the structure of SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, a molecule of water is indeed stabilized between K158 and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, leading to a water bridge formed between K158 and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Therefore, K158 in SenB likely performs an equivalent function as K258 in SelA. The amino side chain of K158 captures the proton from the water molecule, making it a negatively charged nucleophilic agent to attack the positively charged P atoms of Se-P, which leads to the cleavage of the Se-P bond to produce the final product of selenosugar (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). The hypothesized role of K158 during the Se-P bond cleavage was proven by examining the catalyzed products of the functionally impaired SenB mutant K158A (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Using UDP-Glc, UDP-GlcNAc and UDP-GalNAc as sugar donors, the corresponding key selenophosphosugar intermediates were all detected by LC-MS for catalysis by the K158A mutant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). To further consolidate our hypothesis, mutation of K158 to different types of amino acids were introduced and most of the mutants resulted in a complete loss of catalytic activity of SenB. Only the mutants K158R, K158H, and K158N with alkaline side chains or with amino side chains retained weak catalytic activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results further indicated that K158 is indeed involved in the Se-P cleavage and plays an irreplaceable role in the catalytic reaction of SenB. This catalytic mechanism of SenB by catalyzing formation of the C-Se glycosidic bond and cleavage of the Se-P bond in use of the catalytic triad H58/D86/K158 is completely different from that of the other glycosyltransferases that have been reported so far.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eMining and characterization of SenB-like enzymes\u003c/h2\u003e\n\u003cp\u003eWe did gene mining for the homologous SeGTs in the NCBI database using SenB as a probe, and more than 200 SenB-like genes were found (\u0026gt;\u0026thinsp;55% similarity), which were mainly distributed in \u0026beta;-proteobacteria. Sequence conservation analysis of these SenB-like enzymes revealed that the putative catalytic triad H58/D86/K158 exhibit a high degree of conservation, which further supports our hypothesis on the catalytic mechanism of SenB (\u003cstrong\u003eExtended Data Fig.\u0026nbsp;8\u003c/strong\u003e). Besides, there is a highly conserved \u0026ldquo;EGGAHV\u0026rdquo; motif found related to sugar donor binding in SenB-like enzymes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg \u003cstrong\u003eand Extended Data Fig.\u0026nbsp;7\u003c/strong\u003e). One of the three key amino acids that determines the sugar donor promiscuity of SenB, E231, is located within this conserved motif. The other two key amino acids, N20/T23, are also highly conserved among these SenB-like enzymes \u003cstrong\u003e(Extended Data Fig.\u0026nbsp;7)\u003c/strong\u003e. Therefore, we suspect that these SenB-like enzymes may all have a certain degree of sugar donor promiscuity. We selected two SenB-like enzymes, \u003cem\u003eCb\u003c/em\u003eSenB (GenBank: RYF17368.1; 65.5% sequence similarity) derived from \u003cem\u003eComamonadaceae bacterium\u003c/em\u003e and RsSenB (GenBank: MBC7468551.1, 65.2% sequence similarity) derived from \u003cem\u003eRamlibacter sp.\u003c/em\u003e, for further functional characterization \u003cstrong\u003e(Extended Data\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef \u003cstrong\u003eand Supplementary Table\u0026nbsp;3)\u003c/strong\u003e. Sequence alignment showed that \u003cem\u003eCb\u003c/em\u003eSenB and \u003cem\u003eRs\u003c/em\u003eSenB contain the putative catalytic triad H58/D86/K158 as well as the key EGGAHV motif (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Catalytic activity analysis showed that \u003cem\u003eCb\u003c/em\u003eSenB and \u003cem\u003eRs\u003c/em\u003eSenB recognize sugar donors similarly to SenB, and that both utilize UDP-Glc, UDP-GlcNAc, UDP-Gal, and UDP-GalNAc to generate the corresponding selenosugar (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). The catalytic activities of these three enzymes for UDP-Glc, UDP-GlcNAc, and UDP-Gal were similar, but \u003cem\u003eRs\u003c/em\u003eSenB utilized UDP-GalNAc with a stronger capacity than SenB and \u003cem\u003eCb\u003c/em\u003eSenB.\u003c/p\u003e\n\u003cp\u003eTo further confirm our hypothesis on the catalytic mechanism of \u003cem\u003eSe\u003c/em\u003e-glycosylation, we solved the crystal structure of \u003cem\u003eRs\u003c/em\u003eSenB (PDB ID: 8K5U) in apo form at a resolution of 2.15 \u0026Aring; (\u003cstrong\u003eSupplementary Table\u0026nbsp;4\u003c/strong\u003e). Unlike SenB, the asymmetric unit of the crystal contains two \u003cem\u003eRs\u003c/em\u003eSenB molecules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). The monomer structure of \u003cem\u003eRs\u003c/em\u003eSenB is highly similar to that of SenB, with an RMSD of 0.99 \u0026Aring; for Ca atoms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). The spatial positions of the two residues D86/K158 are highly overlapped compared the structures of SenB and \u003cem\u003eRs\u003c/em\u003eSenB. Despite the spatial position shift of residue H58, both are still located in the center of catalytic activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee). In addition, the mutagenesis results of the catalytic triad in \u003cem\u003eRs\u003c/em\u003eSenB and \u003cem\u003eCb\u003c/em\u003eSenB were consistent with those of SenB (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). These results not only provided two new SeGTs, but also further demonstrated the universal effects of the key amino acids N20/T23/E231 on the promiscuity of sugar donor and the catalytic triad H58/D86/K158 on \u003cem\u003eSe\u003c/em\u003e-glycosylation of SenB.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSenB is the only functional identified SeGT in nature, but its structure and catalytic mechanism are completely unknown. In this work, substrate specificity investigations showed that SenB has sugar donor promiscuity and specificity to utilize six sugar donors in the form of UDP. Structures of SenB in complex with four sugar donors were solved including SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, SenB/UDP-GlcNAc, SenB/UDP-GalNAc and UDP-Rha. Structural analysis reveals that loss of π-π interactions, leading to increased steric hindrance in the neighborhood of uracil, is the major factor in the specific recognition of UDP-form sugar donors by SenB. Structural comparison and mutagenesis revealed the critical amino acids N20, T23, and E231 that control sugar donor promiscuity of SenB. Furthermore, catalytic mechanism investigations identified the catalytic triad H58/D86/K158 in SenB that are involved in the formation of the C-Se bond and the cleavage of Se-P, and the catalytic process of \u003cem\u003eSe\u003c/em\u003e-glycosylation was deduced. Finally, two new SeGTs, \u003cem\u003eRs\u003c/em\u003eSenB and \u003cem\u003eCb\u003c/em\u003eSenB were mined, and functionally and structurally characterized. These results revealed the structure of SenB, and the possible mechanism of \u003cem\u003eSe\u003c/em\u003e-glycosylation, providing insights into the diversity of C-Se bond formation as well as Se-P bond cleavage in nature, and theoretical guidance for structure-based engineering modification of SeGT for selenosugars or drugs synthesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staffs from BL19U1 beamlines of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for their assistance during data collection, and Prof B.Y from Yangzhou University for providing selenoglucose as a control. This work was supported by grant to F.L. from the National Key Research and Development Program of China (2021YFA0909500), grant to W.H. from National Natural Science Foundation of China (Grant No. 82304333 to W.H.). and grant to F.L. from the Fundamental Research Funds for the Central Universities (2042019kf0185).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.H. and F.L. designed the experiments. J.S., W.H. and T.X. performed the in vitro enzymatic analysis. W.H., J.S. and Y.H. performed crystallization experiments. W.H. and J.S. analyzed the data. W.H. drafted the manuscript. Z.D. and F.L. revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic coordinates and structure factors of SenB and \u003cem\u003eRs\u003c/em\u003eSenb have been deposited in the Protein Data Bank (www.rcsb.org) with accession codes 8JJT, 8JJQ, 8JJN, 8JK7, and 8K5U, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoyd, R., Selenium stories. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 570 (2011).\u003c/li\u003e\n\u003cli\u003eReich, H. J. \u0026amp; Hondal, R. J. Why Nature Chose Selenium. \u003cem\u003eACS Chem. 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Hieff Clone Plus Multi One Step Cloning Kit was purchased from Yeasen Biotechnology (Shanghai, China). Restriction Enzymes was purchased from New England BioLabs (Ipswich, MA, USA). Codon-optimized gene fragments were purchased from Tsingke (Beijing, China). All crystallization materials and reagents were purchased from Hampton Research (Laguna Niguel, CA, USA). Acetonitrile and formic acid of HPLC grade were purchased from Thermo Fisher Scientific (Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction for protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protein coding DNA sequences were codon-optimized for expression in \u003cem\u003eE. coli\u003c/em\u003e, and commercially synthesized. The DNA fragments were then amplified using Super-Fidelity DNA Polymerase, and ligated with the \u003cem\u003eNde\u003c/em\u003eI and \u003cem\u003eHin\u003c/em\u003edIII linearized pET28a vector using one step cloning kit following the manufacturer\u0026rsquo;s instructions. Ligation mixtures were transformed into chemically competent \u003cem\u003eE. coli\u003c/em\u003e TOP10 by heat shock and plated onto LB agar containing 50\u0026thinsp;\u0026mu;g/mL kanamycin. Single colony was picked and cultured for plasmid extraction. The constructions confirmed by Sanger sequencing were later used for protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSite-Directed Mutagenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSite-directed mutagenesis of SenB was performed using the polymerase chain reaction (PCR) with primers designed to generate the desired mutations (\u003cstrong\u003eSupplementary Table5\u003c/strong\u003e). The wild-type pET28a-SenB plasmid was used as the PCR template. PCR was set up with a Phanta Max Super-Fidelity DNA Polymerase in a Biorad C1000 Thermal Cycler. After digestion of the template DNA with \u003cem\u003eDpn\u003c/em\u003eI for 2 h in 37\u0026deg;C, the PCR products were transformed into the \u003cem\u003eE. coli\u003c/em\u003e TOP10 competent cell. All of the mutations were confirmed by the Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of SenB, SenC, \u003cem\u003eRs\u003c/em\u003eSenB, \u003cem\u003eCb\u003c/em\u003eSenB and mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll proteins were individually produced in \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) cells. Cells transformed with corresponding expression plasmids were cultured in 1 L of LB medium (1% Tryptone, 0.5% Yeast extract, 1% NaCl) containing 50 \u0026micro;g/mL kanamycin, shaken at 220 rpm and 37\u0026deg;C until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6, then induced with 0.4 mM isopropyl \u0026beta;-D-1-thiogalactopyranoside (IPTG) and growth at 18\u0026deg;C for 18 h. Cells were pelleted by centrifugation (SORVALL LYNX 4000, Thermo Scientific) at 6,000 rpm for 10 min, resuspended in 80 mL of lysis buffer consisting of 20 mM Tris-HCl, 300 mM NaCl, pH 7.5, and disrupted by a high-pressure homogenizer (EmulsiFlex-C3, AVESTIN, Canada) at 12,000 psi. The lysate was clarified by centrifugation at 15,000g for 45 min, and the supernatant was loaded onto a gravity column pre-equilibrated in lysis buffer with 5 mL Ni-NTA affinity resins (GenScript, Nanjing, China). The column was washed sequentially with lysis buffer containing 20 mM imidazole for 10 CV (column volume), 50 mM imidazole for 6 CV, and then the target proteins were eluted with lysis buffer containing 300 mM imidazole for 3 CV. The eluted proteins were further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column (GE Healthcare) in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM dithiothreitol (DTT). The peak fractions containing target protein were collected and examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the purified protein was concentrated to 7.5 mg/mL using an Amicon Ultra-30 K filter (Millipore), flash-frozen in the liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for later use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme activity assay of SenB, RsSenB, CbSenB and mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder anaerobic conditions, the reactions were performed in a final volume of 50 \u0026micro;L, containing 20 \u0026micro;M SenC, 20 \u0026micro;M SenB, \u003cem\u003eRs\u003c/em\u003eSenB, \u003cem\u003eCb\u003c/em\u003eSenB or mutants, 2 mM DTT, 2 mM ATP, 1 mM Na\u003csub\u003e2\u003c/sub\u003eSe, and 2 mM of UDP-sugar. All of materials were prepared in buffer consisting of 50 mM Tri-HCl, 20 mM KCl and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.2. Experimental reactions were prepared in an identical manner, except that different mutants of SenB and UDP-sugar were used. After a 6 h incubation period at room temperature, reactions were removed from the glovebox and exposed to atmosphere for 30 min to oxidize any unreacted Na\u003csub\u003e2\u003c/sub\u003eSe. The reactions were then quenched with 50 \u0026micro;L of 10 mM ice cold mBBr in MeCN, followed by incubation in dark at room temperature for an additional 30 min to allow the completeness of derivatization with mBBr. Finally, the supernatants were collected after centrifugation at 12,000 rpm for 30 min, and analyzed by HPLC-UV/DAD and HPLC-MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC and LC\u0026thinsp;\u0026minus;\u0026thinsp;MS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPLC analysis was performed on a Shimadzu-LC-20AT (Japan) with an Ultimate\u0026reg; XB-C18 column (4.6 mm\u0026times;250 mm I.D., 5 \u0026micro;m, Welch Materials, Inc., China) at a flow rate of 0.8 mL/min, using the mobile phase of (A) 0.1% formic acid in deionized H\u003csub\u003e2\u003c/sub\u003eO and (B) 100% MeCN. The gradient settings for separating the products and substrates were 0\u0026thinsp;\u0026minus;\u0026thinsp;20 min 10% B to 50% B, 20\u0026thinsp;\u0026minus;\u0026thinsp;25 min 50% B to 100% B, 25\u0026thinsp;\u0026minus;\u0026thinsp;28 min 100% B to 10% B, and 28\u0026thinsp;\u0026minus;\u0026thinsp;35 min 10% B to 10% B. The products were further confirmed using an LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) The MS/MS analysis was carried out in a positive ionization mode with 35% relative collision energy. The relative activities of the mutants were determined by HPLC and calculated by the product's peak area dividing the wild-type's peak area. All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscale thermophoresis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe purified SenB protein was exchanged into test buffer consisting of 25 mM Hepes, 150 mM NaCl, 0.05% Tween 20, 1 mM DTT, pH 7.5 for the microscale thermophoresis (MST) experiments. The protein was diluted to a final concentration of 1 \u0026micro;M in test buffer, then a 100 nM MO-L018 RED-tris-NTA dye solution was added to protein solution. The protein and dye mixture were mixed well and incubated at 4\u0026deg;C in the dark for 30 min. The labeled protein was obtained by centrifugation at 12,000 rpm for 10 min. The binding affinities between substrates and proteins were analyzed on a Monolith NT.115 instrument (Nanotemper Technologies). Different concentrations of UDP-sugars were serially diluted from the stocks using test buffer (the premade stocks were 40 \u0026micro;M UDP-Glc, 50 \u0026micro;M UDP-Gal, 10 \u0026micro;M UDP-GlcNAc, 20 \u0026micro;M UDP-GalNAc, 1 mM UDP-Rha, and 10 mM UDP-Xyl, respectively). The equal volumes of labeled protein were added to various concentrations of UDP-sugar solutions in a final volume of 10 \u0026micro;L. After being incubated at 4\u0026deg;C in the dark for 30 min, the reaction mixtures were loaded into standard treated capillaries (Monolith NT.115 series capillaries MO-K022) and analyzed by MST at medium MST power and auto-detect excitation power with a laser-on time of 2.5 s and a detection temperature of 25\u0026deg;C. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values were calculated using MO. Affinity Analysis Software from three independent thermophoresis measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelenophosphosugar intermediate analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder anaerobic conditions, enzyme activity assays of the K158A mutant of SenB were performed in an identical manner, except being supplied with different UDP-sugars (UDP-Glc/UDP-GlcNAc/UDP-GalNAc). After a 6 h incubation period at room temperature, reactions were removed from the glovebox and exposed to atmosphere for 30 min to oxidize any unreacted Na\u003csub\u003e2\u003c/sub\u003eSe. The reactions were then quenched with 50 \u0026micro;L MeCN, and centrifuged at 12,000 rpm for 30 min. The supernatants were analyzed by LC-MS. The LC was performed with an COSMOSIL\u0026reg; PBr column (4.6 mm\u0026times;250 mm I.D.) at a flow rate of 0.3 mL/min, using the mobile phase of (A) 10 mM NH\u003csub\u003e4\u003c/sub\u003eAc in deionized H\u003csub\u003e2\u003c/sub\u003eO and (B) 100% MeCN. The Elution settings were 0\u0026thinsp;\u0026minus;\u0026thinsp;25 min 5% B. The products were further confirmed using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein crystallization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe purified SenB protein (7.5 mg/mL) was incubated with UDP-Glc (5mM), UDP-GlcNAc (5 mM), UDP-GalNAc (5 mM), UDP-Rha (5 mM) for 30 min on ice, and the purified \u003cem\u003eRs\u003c/em\u003eSenB protein (7.5 mg/mL) was incubated with UDP-GalNAc (5mM), before set-up of the crystallization trays. Crystals of SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e were observed at 18 ℃ within 3\u0026ndash;4 d using the hanging drop vapor diffusion method by mixing 0.8 \u0026micro;L of protein with 0.8 \u0026micro;L of the reservoir solution (0.2 M Ammonium sulfate, 0.1 M Hepes (pH 7.5), and 20% (w/v) polyethylene glycol 8000, 10% (v/v) 2-Propanol). The crystals of SenB/UDP-GalNAc were obtained in the reservoir solution containing 0.1 M Bis-Tris (pH 6.5), and 20% (w/v) polyethylene glycol 5000-MME. The crystals of SenB/UDP-GlcNAc and SenB/UDP-Rha were obtained in the reservoir solution containing 0.1 M Hepes (pH 7.0), and 15% (w/v) polyethylene glycol 20000. The crystals of \u003cem\u003eRs\u003c/em\u003eSenB/UDP-GalNAc were obtained in the reservoir solution containing 0.2 M Ammonium citrate dibasic, and 20% (w/v) polyethylene glycol 3350. All crystals were harvested in the same reservoir solution supplemented with 20% (w/v) glycerol as the cryo-protectant and flash-frozen in the liquid nitrogen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and structure determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe crystallographic data sets were collected on the beamlines 19U1 at the Shanghai Synchrotron Radiation Facility (SSRF)\u003csup\u003e43\u003c/sup\u003e. The diffraction images were processed using XDS\u003csup\u003e44\u003c/sup\u003e. The structures of SenB and \u003cem\u003eRs\u003c/em\u003eSenB were solved by molecular replacement using a Phaser from the CCP4 suite\u003csup\u003e45\u003c/sup\u003e, and the alphafold2\u003csup\u003e46\u003c/sup\u003e predicted structure of SenB was used as the searching model. The models of the SenB complexes were built initially using AutoBuild\u003csup\u003e47\u003c/sup\u003e and manually using Coot\u003csup\u003e48\u003c/sup\u003e. The iterative refinement and structure validation were done using Phenix\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural visualization analysis and figure preparation were made with Protein-ligand interaction profiler\u003csup\u003e50\u003c/sup\u003e and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schr\u0026ouml;dinger, LLC). Sequence alignments were created using Clustal Omega\u003csup\u003e51\u003c/sup\u003e, ESPript\u003csup\u003e52\u003c/sup\u003e, and WebLogo\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutodock 4.0\u003csup\u003e54\u003c/sup\u003e was used to build the structures of SenB/UDP-Xyl, SenB/UDP-Gal, and SenB/UDP-Glc/SeP, using SenB/UDP-Glc/PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e as the template.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e43 Zhang, W.-Z. \u003cem\u003eet al.\u003c/em\u003e The protein complex crystallography beamline (BL19U1) at the Shanghai Synchrotron Radiation Facility. \u003cem\u003eNucl. Sci. Tech.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 170 (2019).\u003c/p\u003e\n\u003cp\u003e44 Kabsch, W. Xds. \u003cem\u003eActa Crystallogr. D Biol. Crystallogr.\u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 125\u0026ndash;132 (2010).\u003c/p\u003e\n\u003cp\u003e45 Bunkoczi, G. \u003cem\u003eet al.\u003c/em\u003e Phaser.MRage: automated molecular replacement. \u003cem\u003eActa Crystallogr. D Biol. Crystallogr.\u003c/em\u003e\u003cstrong\u003e69\u003c/strong\u003e, 2276\u0026ndash;2286 (2013).\u003c/p\u003e\n\u003cp\u003e46 Jumper, J. \u003cem\u003eet al.\u003c/em\u003e Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e596\u003c/strong\u003e, 583\u0026ndash;589 (2021).\u003c/p\u003e\n\u003cp\u003e47 Terwilliger, T. C. \u003cem\u003eet al.\u003c/em\u003e Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. \u003cem\u003eActa Crystallogr. D Biol. Crystallogr.\u003c/em\u003e\u003cstrong\u003e64\u003c/strong\u003e, 61\u0026ndash;69 (2008).\u003c/p\u003e\n\u003cp\u003e48 Emsley, P., Lohkamp, B., Scott, W. G. \u0026amp; Cowtan, K. Features and development of Coot. \u003cem\u003eActa Crystallogr. D Biol. Crystallogr.\u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 486\u0026ndash;501 (2010).\u003c/p\u003e\n\u003cp\u003e49 Adams, P. D. \u003cem\u003eet al.\u003c/em\u003e PHENIX: a comprehensive Python-based system for macromolecular structure solution. \u003cem\u003eActa Crystallogr. D Biol. Crystallogr.\u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 213\u0026ndash;221 (2010).\u003c/p\u003e\n\u003cp\u003e50 Adasme, M. F. \u003cem\u003eet al.\u003c/em\u003e PLIP 2021: expanding the scope of the protein-ligand interaction profiler to DNA and RNA. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e\u003cstrong\u003e49\u003c/strong\u003e, W530-W534 (2021).\u003c/p\u003e\n\u003cp\u003e51 Madeira, F. \u003cem\u003eet al.\u003c/em\u003e The EMBL-EBI search and sequence analysis tools APIs in 2019. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e\u003cstrong\u003e47\u003c/strong\u003e, W636-W641 (2019).\u003c/p\u003e\n\u003cp\u003e52 Robert, X. \u0026amp; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e\u003cstrong\u003e42\u003c/strong\u003e, W320-324 (2014).\u003c/p\u003e\n\u003cp\u003e53 Crooks, G. E., Hon, G., Chandonia, J. M. \u0026amp; Brenner, S. E. WebLogo: a sequence logo generator. \u003cem\u003eGenome Res\u003c/em\u003e. \u003cstrong\u003e14\u003c/strong\u003e, 1188\u0026ndash;1190 (2004).\u003c/p\u003e\n\u003cp\u003e54 Trott, O. \u0026amp; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e\u003cstrong\u003e31\u003c/strong\u003e, 455\u0026ndash;461 (2010).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Selenium, Se-glycosyltransferase, Crystal structure, Se-glycosylation, Catalytic mechanism","lastPublishedDoi":"10.21203/rs.3.rs-3357770/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3357770/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSelenium is a multi-functional trace element essential for diverse organisms. SenB is a \u003cem\u003eSe\u003c/em\u003e-glycosyltransferase that incorporates selenium into small molecules in the selenoneine biosynthesis pathway and is also the only known \u003cem\u003eSe\u003c/em\u003e-glycosyltransferase in nature. Although the biochemical function of SenB has been investigated, its substrate specificity, structure, and catalytic mechanism remain unclear. Here, we revealed that SenB exhibits sugar donor specificity and promiscuity and can utilize six UDP-sugars to generate selenosugars. The crystal structures of SenB complexed with four different UDP-sugars were solved. The residues N20, T23, and E231 were proven as the key elements that determine the sugar donor promiscuity of SenB. Structure-guided mutagenesis further revealed a novel catalytic triad H58/D86/K158 in SenB, which accounts for the C-Se glycosidic bond formation and Se-P bond cleavage during the \u003cem\u003eSe\u003c/em\u003e-glycosylation process. Furthermore, we mined, functionally and structurally characterized two other novel \u003cem\u003eSe\u003c/em\u003e-glycosyltransferase, \u003cem\u003eCb\u003c/em\u003eSenB and \u003cem\u003eRs\u003c/em\u003eSenB, which also exhibit sugar donor promiscuity.\u003c/p\u003e","manuscriptTitle":"Structural basis for substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-28 15:40:58","doi":"10.21203/rs.3.rs-3357770/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ed6a73dc-9122-426f-ad73-475f50d9b873","owner":[],"postedDate":"September 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":25018779,"name":"Biological sciences/Chemical biology/Enzyme mechanisms"},{"id":25018780,"name":"Biological sciences/Structural biology/X-ray crystallography"},{"id":25018781,"name":"Biological sciences/Chemical biology/Enzymes"}],"tags":[],"updatedAt":"2024-02-24T08:07:58+00:00","versionOfRecord":{"articleIdentity":"rs-3357770","link":"https://doi.org/10.1038/s41467-024-46065-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-02-23 05:00:00","publishedOnDateReadable":"February 23rd, 2024"},"versionCreatedAt":"2023-09-28 15:40:58","video":"","vorDoi":"10.1038/s41467-024-46065-6","vorDoiUrl":"https://doi.org/10.1038/s41467-024-46065-6","workflowStages":[]},"version":"v1","identity":"rs-3357770","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3357770","identity":"rs-3357770","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}