Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid

Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid Hongxiu Liao, Haifeng Pan, Jinfeng Yao, Ronglin Zhu, Wenna Bao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3437865/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 May, 2024 Read the published version in Biotechnology Letters → Version 1 posted 5 You are reading this latest preprint version Abstract Objectives This study aimed to discuss the essential amino acid residues and catalytic mechanism of trans -epoxycussinate hydrolase from Pseudomonas koreensis for production of meso -tartaric acid. Results The optimum conditions of the enzyme were 45°C and pH 9.0, respectively. It was strongly inhibited by Zn 2+ , Mn 2+ and SDS. Michaelis-Menten enzyme kinetics analysis gave a K m value of 3.50 mM and a k cat of 99.75 s − 1 , the EE value was higher than 99.9%. Multiple sequence alignment and homology modeling showed that the enzyme belonged to MhpC superfamily and had a typical α/β hydrolase folding structure. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272 were important catalytic residues. 18 O-labeling study suggested the enzyme acted via two-step catalytic mechanism. Conclusions The structure and catalytic mechanism of trans -epoxycussinate hydrolase were firstly reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad were proposed. trans-epoxysuccinate hydrolase Pseudomonas koreensis meso-tartaric acid catalytic mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Epoxysuccinate hydrolases (ESHs) are members of epoxide hydrolases (EHs, EC 3.3.2.3) that catalyze the epoxysuccinate to tartaric acid (TA). The enzymatic properties of ESHs determine the stereospecificity of TA. TA has three optically active isomers including L (+)-TA, D (-)-TA and meso -TA, and their corresponding ESHs are named ESH( L ), ESH( D ) and ESH( meso ), respectively (Xuan et al. 2019). meso -TA is a rare enantiomer of TA. It is described the most preferred anti-caking agent, has the advantage of environmentally safe, non-toxic, effective in small amounts, etc. meso -TA has great market potential for chloride production and snow removal. In addition, it is also used in pharmaceuticals, ani-malaria and new materials. The demand of meso -TA is increasing yearly (Varga et al. 2013 ; Dutta et al. 2017; Bitew et al. 2019 ). At present, the conversion efficiency of TA using chemical synthesis method is low due to the difficulty in separation of different configurations of TA. Biotransformation has the additional advantages, such as mild reaction conditions; excellent chemo-, regio-, and enantio-selectivity, is considered to be a simple and economical way to produce TA (Bao et al. 2020 ). The protein structures and catalysis mechanisms of ESH( L ) (Pan et al. 2011 ; Cheng et al. 2014a ) and ESH( D ) (Bao et al. 2013 ; Dong et al. 2018 ) have been reported. However, a few ESHs( meso ) have been reported. In 1955, Martin and Foster firstly screened Flavobacterium sp. which can transform trans -epoxysuccinate to meso -TA, and named this enzyme as trans - epoxysuccinate hydrolase (TESH) (Martin et al. 1955; Foster 1960 ). In 1969, Allen and Jakoby ( 1969 ) purified TESH from Pseudomonas putida , and studied its enzymatic properties. Recently, Pseudomonas koreensis BK-9 was isolated to produce meso -TA and its TESH gene was cloned in our lab. However, there have been no further reports on the structure and catalytic mechanism of the TESH. In this study, we characterized the enzymatic properties of TESH from P. koreensis BK-9, investigated its structure, isolated critical amino acid and proposed its catalytic mechanism through multiple sequence alignment, homologous modeling, molecular docking, site-directed mutagenesis and isotope labeling. The results showed that ten residues (H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272) were critical for catalysis and we proposed a two-step mechanism by an Asp-His-Asp catalytic triad. Materials and methods Strains, plasmids, primers, and culture conditions P. koreensis BK-9 was isolated by our lab and deposited at the China General Microbiological Culture Collection Center (identification no: CGMCC NO.8395). The BK-9 strain was cultivated according to the method described by Bao and coworkers ( 2013 ). Its TESH gene was cloned and expressed in Escherichia coli BL21 (DE3) -pET-15b (+) -TESH by our laboratory. Multiple sequence alignment and homology modeling The amino acid sequence of TESH from P. koreensis BK-9 was aligned by Clustal Omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ), to screen conserved sites. The modeled TESH was generated by Swiss-Model ( https://swissmodel.expasy.org/ ). The docking of the trans -epoxysuccinate with the modeled TESH structure was studied with Discovery Studio 2019 client, and the docking results was visualized by PyMol (Version 2.5). Construction of site-directed mutagenesis Based on the reaction mechanism study of the ESH and the results of the alignment, site-directed mutagenesis was carried out using the PrimeSTAR ® Max DNA Polymerase (Takara) with two reverse complement primers. The primers shown in Table S1 were synthesized by Sangon Biotech (Shanghai, China). Enzyme purification and characterization Both wild-type and mutant TESHs were expressed in E.coli BL21 (DE3) cells. The fractions containing TESH were pooled by His-binding resin column (York Biotech, Shanghai, China) (Bao et al. 2013 ). The salt ions were removed by ultrafiltration and enzyme solution was used to investigate the effect of temperature, pH, metal ions and surfactants on the activity and stability of TESH (Wang et al. 2012 ). The K m , V max and k cat values of the enzymes were determined by means of Michaelis-Menten plots at increasing trans -epoxysuccinate concentrations ranging from 5 to 170 mM. Isotope label The synthesis of TA requires the participation of water molecules, we use H 2 18 O to label the reaction system. For single turnover reaction, 200 nmol of TESH and 40 nmol disodium trans -epoxysuccinate were dissolved in 500 µL of H 2 18 O, then incubated the mixture at 30°C for 12 h. For multiple turnover experiment, 4 nmol of TESH and 400 nmol disodium trans -epoxysuccinate were mixed in 500 µL of H 2 18 O and incubated at 30°C for 12 h. The reaction is terminated with an equal volume of methanol, then measured the molecular mass of the produced tartrate by G6465B Ultivo Triple Quadrupole LC/MS (Agilent, USA) (Bao et al. 2013 ). Enzyme assay TESH activity was assay at 37°C for 30 min in 1 mL of 0.2 M disodium trans -epoxysuccinate (pH 8.0). The quantity of meso -TA was determined by Chirex 3126 (D)-penicillanmin (50×4.6 mm) (Cheng et al. 2014b ). One unit of ESH activity was defined as the amount of the enzyme that generating 1µmol meso -TA per minute. Specific activity was defined as the number of units per milligram protein. Protein was determined by the Bradford protein assay kit (Shanghai, China). Results and discussion Characterization of TESH The optimum temperature and pH of TESH from P. koreensis BK-9 were 45°C and pH 9.0, respectively (Fig. 1 A, 1 B). However enzyme activity was almost lost when incubated at 45°C for 30 min, only 10% of residual activity remained when pH was lower than 5 or pH was higher than 10 (Fig. 1 A, 1 C), indicating poor temperature and pH stability. Insert Fig. 1 Statistical analysis indicated that Mg 2+ , Ca 2+ were the activators of the enzyme, increased TESH activity significantly (P ＜ 0.001). Zn 2+ and Mn 2+ had strongly inhibited the activity of TESH ( P＜ 0.001). The addition of EDTA reduced the activity of the enzyme by 46.63%. SDS was a strong inhibitor, decreasing 95.85% activity of the enzyme (Fig. 1 D). The kinetic parameters were determined by Michaelis-Menten plotting method (GraphPad Prism 9.5). The vales of K m and k cat were calculated to be 3.50 mM and 94.75 s − 1 , respectively (Fig. S1). Sequence comparison of TESH and reported EHs The sequence alignment results indicated that TESH shows no homology to other reported ESHs (ESH( L ) belongs to HAD like superfamily, ESH( D ) belongs to BKACE superfamily) that belongs to MhpC superfamily. We found several structurally characterized proteins with low but considerable sequence identity (between 19% and 46%) were shown in a Clustal Omega (Fig. 2 ). Several residues were highly conserved: nine nonpolar amino acids (G23, G25, G33, G61, G63, L48, L59, A49, P235), two arginine (D58 and D104), two histidine (H103 and H272) and one lysine (Y211), polar amino acids play an important role in enzyme catalysis. Besides, E35 and H104 form ion pairs in StEH1, so we also designed corresponding Q36 mutated amino acid in TESH. Insert Fig. 2 Homology modeling of TESH We selected 4NVR as the template structure to generate the 3D structure of the TESH from P. koreensis BK-9 with Swiss-Model, this template possesses the greatest amino acid sequence identity with TESH (46.52%). The root mean square deviation value of the modeled TESH was 0.068 Å (Fig. S2). The Ramachandran plot (Fig. S3) showed that the percentages of residues falling in disallowed regions, generously allowed regions, favorable regions and core regions were 0, 1.2, 9.3, and 89.5%, respectively. An overall plot showed that more than 95% of the residues were within the favorable region, which revealed that the quality of the model was good. The modeled 3D structure of TESH has the typical eight parallel β-sheets (residues 6–10, 16–22, 27–31, 53–57, 98–103, 121–127, 236–241 and 263–268) surrounded by α-helices and is covered by a cap-domain (Fig. 3 A). It is similar with the structure from MhpC superfamily (StEH1, AD1, CorEH, and FAcD). Insert Fig. 3 The trans -epoxysuccinate substrate was docked in the core of the modeled TESH structure, results showed that there were thirteen amino acids (H34, D104, R105, R108, D128, I129, Y147, H149, W150, V175, R179, Y211, H272) near the binding pocket, which were labeled on the modeled structure by PyMOL (Fig. 3 B). Site-directed mutagenesis Combing the results of multiple sequence alignment and molecular docking, we selected fourteen amino acid sites to replaced their polar amino and conserved charged acid residues with another residues as follows: H and D by N; Q by E; R by K; I, V and W by A; Y by F. Purified protein were showed in Fig. S4. The results of site-directed mutagenesis revealed that six mutants showed relative activities that were only 2% or less of the wild-type enzyme’s activity: R108K, D128N, Y147F, H149N, W150A and Y211F. Four mutants showed the lower k cat / K m value than the wild-type: H34N, D104N, R105K, Y211F (Table 1 ). The results revealed that the ten residues (H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272) played important roles in the catalysis. Table 1 Characterization of wild-type and mutant TESH enzyme. a Enzyme Relative activity(%) K m (mM) k cat (s − 1 ) k cat / K m (mM − 1 s − 1 ) Wile-type 100.00 ± 2.34 3.50 94.75 27.08 H34N 5.75 ± 0.39 7.88 4.73 0.60 Q36E 13.40 ± 0.67 2.80 50.41 18.02 D104N 54.53 ± 0.96 83.75 11.41 0.14 R105K 7.94 ± 0.26 17.53 9.06 0.52 R108K 0.20 ± 0.06 - - - D128N 0.53 ± 0.04 - - I129A 8.64 ± 0.83 10.48 21.25 2.03 Y147F 1.81 ± 0.09 - - - H149N 0.34 ± 0.00 - - - W150A 0.55 ± 0.12 - - - V175A 20.79 ± 0.40 6.93 16.88 2.44 R179K 19.04 ± 0.60 13.20 22.09 1.67 Y211F 0.15 ± 0.03 2.82 0.42 0.15 H272N 11.21 ± 0.20 3.78 15.39 4.07 a The specific activity of the wild-type was 124.38 ± 2.34 µmol min − 1 mg − 1 . Insert Table 1 Single and multiple turnover reaction of wild-type TESH in H 2 18 O The single and multiple turnover reactions of TESH from P. koreensis BK-9 in H 2 18 O showed that most of the tartrate was 16 O-labeled in the single turnover reaction (Fig. 4 A), while most of the tartrate was 18 O-labeled in the multiple turnover reaction (Fig. 4 B). These results suggested that the catalytic mechanism of the TESH from P. koreensis BK-9 same as most α/β hydrolases that proceeded by a two-step catalytic reaction involving the forming of an enzyme-substrate ester intermediate. Insert Fig. 4 The ten important catalytic residues of the TESH we obtained were aligned with the dehalogenase from R. palustris CGA009 (FAcD), epoxide hydrolase from Corynebacterium sp. C12 (CorEH), epoxide hydrolase from A. radiobacter (AD1), epoxide hydrolase from S. tuberosum ( StEH1) and CFTR inhibitory factor Cif from P. aeruginosa (Cif). We found that these ten residues were similar to the active sites of the FAcD, CorEH, AD1, StEH1 and Cif (Fig. 5 A), which have a two-step catalytic mechanism characterized by an aspartate-histidine-aspartate catalytic triad (Nardini et al. 1999 ; Chan et al. 2011 ; Bahl et al. 2016 ; Bauer et al. 2016 ; Schuiten et al. 2021 ). Therefore, the TESH may consists of the catalytic nucleophile Asp104, the charge relay acid Asp128 and the histidine base His272 to forming an Asp104-His272-Asp128 catalytic triad, which is shown in Fig. 5 B. Insert Fig. 5 Like most of EHs, Tyr residue that near the substrate plays an important role in help opening the ring. Firstly, they have hydrogen bond with the epoxide oxygen, which can position the substrate in the active site for nucleophilic attack. Then, tyrosine provide a proton to the epoxide oxygen during the ring-opening step (Yamada et al. 2000 ). While epoxide hydrolases usually use Tyr-Tyr pair to assist ring-opening (Nardini et al. 1999 ; Loo et al. 2006; Bauer et al. 2016 ), some epoxide hydrolases use a His-Tyr pair instead (Chan et al. 2011 ; Bahl et al. 2012; Schuiten et al. 2021 ). We propose Tyr211 co-action with Tyr147-His149-Trp150 to open the ring of trans -epoxysuccinate (Fig. 5 C). When nucleophilic Asp104 attack oxirane ring in the opposite side forming enzyme-substrate intermediate, Tyr211 co-action Tyr147-His149-Trp150 to form an oxyanion hole that stabilizes the oxyanion of the alkyl-enzyme intermediate formed by ring opening (Amrein et al. 2015 ). In FAcD Arg111-Arg114 pair are the binding sites for carboxylic acids (Chan et al. 2011 ), in CorEH Trp100 is the halide-stabilizing residue (Schuiten et al. 2021 ), Trp38 and Phe108 have hydrogen bonds with Asp107 thus stabilize the intermediate (Nardini et al. 1999 ). Glu35-His104 ion pair in StEH1 can control solvent access to the active site (Amrein et al. 2015 ). Same as AD1, CFTR inhibitory factor Cif also have hydrogen bond between Phe63 and Ile130 which can stabilize intermediate (Bahl et al. 2016 ). According to homologous molecular docking results, Arg105 and Arg108 from TESH have hydrogen bond with the carboxylate binding site of trans -epoxysuccinate (Fig. 5 D). H34 have polar contacts with D104 and Y211 (Fig. 5 E). We propose like StEH1, H34 in TESH also play the same role as E35 is the part of a hydrogen-bond network that connects the active site with the solvent interface (Amrein et al. 2015 ). More experiments are needed to confirm this conjecture. Based on the results and the above analysis, we speculated that the TESH adopted a two-step catalytic mechanism via Asp104-His272-Asp128 catalytic triad (Fig. 6 ). The catalytic mechanism of ESH( meso ) is similar to that of ESH( L ) from R.opacus and Klebsiella sp. BK-58, which are also proceeded through the two-step mechanism involving the formation of a covalent intermediate (Pan et al. 2011 ; Cheng et al. 2014a ). But it is different from the ESH( D ) from Bordetella sp. BK-52, which catalyzes by means of a Zn 2+ -dependent, one-step mechanism (Dong et al. 2018 ). Insert Fig. 6 Conclusions TESH from P. koreensis BK-9 belonged to MhpC superfamily and had a typical α/β hydrolase folding structure. Enzymatic results showed the optimum conditions of the enzyme were 45°C and pH 9.0, enzyme activity was strongly inhibited by Zn 2+ , Mn 2+ and SDS. Michaelis-Menten constant K m was 3.50 mmol L − 1 , the constant of catalytic activities was 99.75 s − 1 , the enantiomeric purity was higher than 99.9%. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272 were important residues involved in catalysis. Isotope labeling study suggested the enzyme acted via Asp104-His272-Asp128 two-step catalytic mechanism. Declarations Author contributions WNB and HFP contributed to the study conception and design. Material preparation, data collection and analysis were performed by HXL, JFY and RLZ. The first draft of the manuscript was written by HXL and all authors commented on previous version of the manuscript. All authors read and approved the final manuscript. Funding This study was funded by the Huzhou Scientific and Technological Project (2022GZ56) and the Education of Zhejiang Province of China (Y202248484). Conflict of interest All authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study. References Allen RH, Jakoby WB (1969) Tartaric acid metabolism. IX. Synthesis with tartrate epoxidase. 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J Biol Chem 275: 23082-88. http://dx.doi.org/10.1074/jbc.M001464200 Supplementary Files Supportinginformation.docm Cite Share Download PDF Status: Published Journal Publication published 12 May, 2024 Read the published version in Biotechnology Letters → Version 1 posted Reviewers agreed at journal 06 Jan, 2024 Reviewers invited by journal 27 Nov, 2023 Editor assigned by journal 14 Nov, 2023 First submitted to journal 13 Nov, 2023 Editorial decision: Major revisions 20 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-3437865","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":254098893,"identity":"50d78e75-a4a7-454e-b8fc-d46383f94941","order_by":0,"name":"Hongxiu Liao","email":"","orcid":"","institution":"Zhejiang University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongxiu","middleName":"","lastName":"Liao","suffix":""},{"id":254098894,"identity":"e6cc621c-6802-4186-b7df-1e39404e55fc","order_by":1,"name":"Haifeng Pan","email":"","orcid":"","institution":"Huzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haifeng","middleName":"","lastName":"Pan","suffix":""},{"id":254098895,"identity":"4b4d4dbb-76c1-4414-810a-adb796477204","order_by":2,"name":"Jinfeng Yao","email":"","orcid":"","institution":"Zhejiang University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Yao","suffix":""},{"id":254098896,"identity":"f9613921-2d44-4eeb-a822-2252cd55dc13","order_by":3,"name":"Ronglin Zhu","email":"","orcid":"","institution":"Zhejiang University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ronglin","middleName":"","lastName":"Zhu","suffix":""},{"id":254098897,"identity":"40039e52-af50-4f84-b009-f048e4e7cce6","order_by":4,"name":"Wenna Bao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYLACxgYGBn5m5sMPSNMi2c6WZkCaFoPzPAoSRKk2ON577MHPHXZyxod5GAwYamyiCWs5cy7dsPdMsrHZYd4DDxiOpeU2ENJidiPHTIK3jTlx22G+BAPGhsNEaLn/xkzyb1t94uZmHgMJ4rTc4DGT5m07nLiBmVgt9mdyzKRlzxw3ljgMDOQEYvwi2X7GTPLtjmo5/v7Dhx98qLEhrAUVJJCmfBSMglEwCkYBLgAAFuA9ozyxnIMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3452-7938","institution":"Zhejiang University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wenna","middleName":"","lastName":"Bao","suffix":""}],"badges":[],"createdAt":"2023-10-12 14:11:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3437865/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3437865/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10529-024-03490-3","type":"published","date":"2024-05-13T00:41:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":47395055,"identity":"3aa4d20f-eded-446c-884e-8eaf406a5a9b","added_by":"auto","created_at":"2023-11-30 18:59:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":447874,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9. Values are mean±SD calculating from triplicate determinations. The values are shown as relative activities. The activity under the optimal condition is indicated as 100%.\u003cstrong\u003e A\u003c/strong\u003e Effect of temperature on enzyme activity and stability. \u003cstrong\u003eB\u003c/strong\u003e Effect of pH on enzyme activity. \u003cstrong\u003eC\u003c/strong\u003eEffect of pH on enzyme stability. \u003cstrong\u003eD\u003c/strong\u003e Effect of various metal ions and some chemicals on activity of enzyme. (T test, *P ＜ 0.1, **P ＜ 0.01, ***P ＜ 0.001, ****P ＜ 0.0001)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/75d74fedcd9202cc08c40e1b.png"},{"id":47395056,"identity":"65cc19dc-06ef-4242-91ce-2052697bff6c","added_by":"auto","created_at":"2023-11-30 18:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1856509,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignments for TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 and nine structurally characterized proteins from the MhpC superfamily. The sequences were aligned with Clustal Omega and were shown in order of decreasing sequence identity compared to TESH. Identical amino acids are marked with an asterisk, conserved substitution residues with a colon, semiconserved substitution residues with a period, and residues mutagenized in this study with a number sign. α-Helices are underlined, and β-strands are shaded. Sequences: 4NVR, putative acyltransferase from \u003cem\u003eS. enterica \u003c/em\u003e(WP_000105593); 4B9A, putative epoxide hydrolase from \u003cem\u003eP. aeruginosa\u003c/em\u003e (WP_003114850); 3R3Z, fluroacetate dehalogenase RPA1163 from \u003cem\u003eR. palustris\u003c/em\u003e CGA009 (WP_011156727); 6KXR, putative hydrolase Alp 1U from \u003cem\u003eS. ambofaciens\u003c/em\u003e ATCC 23877 (WP_05315812); 7AC0, epoxide hydrolase CorEH from \u003cem\u003eCorynebacterium\u003c/em\u003e sp. C12 (PDB:7AC0_AAA); 3QYJ putative α/β hydrolase from \u003cem\u003eNostoc\u003c/em\u003e sp. PCC 7120 (WP_010994216); 1EHY, Epoxide hydrolase from \u003cem\u003eA. radiobacter \u003c/em\u003eAD1 (PDB:1EHY_A); 2CJP, epoxide hydrolase StEH1 from\u003cem\u003e S. tuberosum\u003c/em\u003e(NP_1275417); 3KD2, CFTR inhibitory factor Cif from \u003cem\u003eP. aeruginosa\u003c/em\u003eUCBPP-PA14 (WP_003452453); and TESH, \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate hydrolase from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/255f15407d6cf54efea0840a.png"},{"id":47395059,"identity":"36a6aa5e-baee-48ed-8230-6fd263eb2f41","added_by":"auto","created_at":"2023-11-30 18:59:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1862220,"visible":true,"origin":"","legend":"\u003cp\u003eHomology modeled structure of TESH. \u003cstrong\u003eA\u003c/strong\u003e, single monomer of TESH highlighting the highly conserved main domain consisting of β-Strands (magenta) surrounded by α-Helices (cyan) and the variable cap domain (orange) typical for members of the α/β hydrolase fold superfamily. The binding pocket is represented by gray. \u003cstrong\u003eB\u003c/strong\u003e, substrate and thirteen amino acids that near the binding pocket are shown in green and yellow respectively\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/51c2186bf714a3d88a2f5d97.png"},{"id":47395061,"identity":"5470b13f-61bb-46c1-a396-d011b0fed365","added_by":"auto","created_at":"2023-11-30 18:59:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":160665,"visible":true,"origin":"","legend":"\u003cp\u003eIon spray mass spectra of tartrate produced by the TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 in H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO. \u003cstrong\u003eA\u003c/strong\u003e, single turnover reaction. \u003cstrong\u003eB\u003c/strong\u003e, multiple turnover reaction. The reaction mixtures were analyzed by system. The step size was 0.1 atomic mass units, and the dwell time was 10 ms per step. The ion spray voltage was set at 4kV, and the orifice voltage was optimized at 50 V (Bao et al. 2013)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/9ffe2fdf2418939c8e663e88.png"},{"id":47395879,"identity":"cd1c7434-e081-4df6-8221-3742c5e53f31","added_by":"auto","created_at":"2023-11-30 19:07:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":729670,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of TESH and comparison of the active site to the numbers of the MhpC superfamily. \u003cstrong\u003eA\u003c/strong\u003e, alignment of the active site of TESH with the dehalogenase from \u003cem\u003eR. palustris\u003c/em\u003eCGA009 (FAcD), epoxide hydrolase from \u003cem\u003eCorynebacterium\u003c/em\u003e sp. C12 (CorEH), epoxide hydrolase from \u003cem\u003eA. radiobacter \u003c/em\u003e(AD1), epoxide hydrolase from\u003cem\u003e S. tuberosum \u003c/em\u003e(StEH1), CFTR inhibitory factor Cif from \u003cem\u003eP. aeruginosa\u003c/em\u003e (Cif), and catalytic residues are highlighted. The catalytic triad is shown with the catalytic nucleophile (red), the charge-relay acid (blue), and the histidine base (purple). The epoxide ring-open pair (green) and substrate/intermediate-stabilizing residues (orange) are also visualized. \u003cstrong\u003eB\u003c/strong\u003e, Asp104-His272-Asp128 catalytic triad of TESH. \u003cstrong\u003eC\u003c/strong\u003e, the residues help epoxide ring-open. \u003cstrong\u003eD\u003c/strong\u003e, the residues can stabilize the substrate/intermediate. \u003cstrong\u003eE\u003c/strong\u003e, the residues that have hydrogen bond with His34\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/43ef11b88dd0d0ab037f2dc3.png"},{"id":47395058,"identity":"b8f7e4f4-944c-4ffb-a917-056960254dc5","added_by":"auto","created_at":"2023-11-30 18:59:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48508,"visible":true,"origin":"","legend":"\u003cp\u003eProposed catalytic mechanism for the TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9. First, S\u003csub\u003eN\u003c/sub\u003e2 attack on the carbon atom in the oxirane ring of the substrate by the nucleophilic Asp104 results in the forming of an enzyme-substrate intermediate jointed by an ester bond; then, the ester bond is hydrolyzed by an active water molecule active by His272, which makes an ion pair with Asp128 leading to the formation of \u003cem\u003emeso\u003c/em\u003e-tartaric acid and the release of the free halide\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/24be085c8cdaed7fa0bb51d6.png"},{"id":56416538,"identity":"d99fbee8-11f8-4e32-ac48-ed2cddf99b05","added_by":"auto","created_at":"2024-05-14 00:41:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3156520,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/965eacd9-0f04-4d35-8aff-400891c62fb2.pdf"},{"id":47395062,"identity":"a857280f-7408-4239-9e49-939c5b747e26","added_by":"auto","created_at":"2023-11-30 18:59:02","extension":"docm","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2800421,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docm","url":"https://assets-eu.researchsquare.com/files/rs-3437865/v1/c5915ee96c9df09d80d9ddde.docm"}],"financialInterests":"","formattedTitle":"Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpoxysuccinate hydrolases (ESHs) are members of epoxide hydrolases (EHs, EC 3.3.2.3) that catalyze the epoxysuccinate to tartaric acid (TA). The enzymatic properties of ESHs determine the stereospecificity of TA. TA has three optically active isomers including \u003cem\u003eL\u003c/em\u003e(+)-TA, \u003cem\u003eD\u003c/em\u003e(-)-TA and \u003cem\u003emeso\u003c/em\u003e-TA, and their corresponding ESHs are named ESH(\u003cem\u003eL\u003c/em\u003e), ESH(\u003cem\u003eD\u003c/em\u003e) and ESH(\u003cem\u003emeso\u003c/em\u003e), respectively (Xuan et al. 2019).\u003c/p\u003e \u003cp\u003e \u003cem\u003emeso\u003c/em\u003e-TA is a rare enantiomer of TA. It is described the most preferred anti-caking agent, has the advantage of environmentally safe, non-toxic, effective in small amounts, etc. \u003cem\u003emeso\u003c/em\u003e-TA has great market potential for chloride production and snow removal. In addition, it is also used in pharmaceuticals, ani-malaria and new materials. The demand of \u003cem\u003emeso\u003c/em\u003e-TA is increasing yearly (Varga et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dutta et al. 2017; Bitew et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt present, the conversion efficiency of TA using chemical synthesis method is low due to the difficulty in separation of different configurations of TA. Biotransformation has the additional advantages, such as mild reaction conditions; excellent chemo-, regio-, and enantio-selectivity, is considered to be a simple and economical way to produce TA (Bao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The protein structures and catalysis mechanisms of ESH(\u003cem\u003eL\u003c/em\u003e) (Pan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e) and ESH(\u003cem\u003eD\u003c/em\u003e) (Bao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) have been reported. However, a few ESHs(\u003cem\u003emeso\u003c/em\u003e) have been reported. In 1955, Martin and Foster firstly screened \u003cem\u003eFlavobacterium\u003c/em\u003e sp. which can transform \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate to \u003cem\u003emeso\u003c/em\u003e-TA, and named this enzyme as \u003cem\u003etrans\u003c/em\u003e- epoxysuccinate hydrolase (TESH) (Martin et al. 1955; Foster \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). In 1969, Allen and Jakoby (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1969\u003c/span\u003e) purified TESH from \u003cem\u003ePseudomonas putida\u003c/em\u003e, and studied its enzymatic properties. Recently, \u003cem\u003ePseudomonas koreensis\u003c/em\u003e BK-9 was isolated to produce \u003cem\u003emeso\u003c/em\u003e-TA and its TESH gene was cloned in our lab.\u003c/p\u003e \u003cp\u003eHowever, there have been no further reports on the structure and catalytic mechanism of the TESH. In this study, we characterized the enzymatic properties of TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9, investigated its structure, isolated critical amino acid and proposed its catalytic mechanism through multiple sequence alignment, homologous modeling, molecular docking, site-directed mutagenesis and isotope labeling. The results showed that ten residues (H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272) were critical for catalysis and we proposed a two-step mechanism by an Asp-His-Asp catalytic triad.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, plasmids, primers, and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 was isolated by our lab and deposited at the China General Microbiological Culture Collection Center (identification no: CGMCC NO.8395). The BK-9 strain was cultivated according to the method described by Bao and coworkers (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Its TESH gene was cloned and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) -pET-15b (+) -TESH by our laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMultiple sequence alignment and homology modeling\u003c/h2\u003e \u003cp\u003eThe amino acid sequence of TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 was aligned by Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to screen conserved sites. The modeled TESH was generated by Swiss-Model (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The docking of the \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate with the modeled TESH structure was studied with Discovery Studio 2019 client, and the docking results was visualized by PyMol (Version 2.5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of site-directed mutagenesis\u003c/h2\u003e \u003cp\u003eBased on the reaction mechanism study of the ESH and the results of the alignment, site-directed mutagenesis was carried out using the PrimeSTAR\u003csup\u003e\u0026reg;\u003c/sup\u003e Max DNA Polymerase (Takara) with two reverse complement primers. The primers shown in Table S1 were synthesized by Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme purification and characterization\u003c/h2\u003e \u003cp\u003eBoth wild-type and mutant TESHs were expressed in \u003cem\u003eE.coli\u003c/em\u003e BL21 (DE3) cells. The fractions containing TESH were pooled by His-binding resin column (York Biotech, Shanghai, China) (Bao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The salt ions were removed by ultrafiltration and enzyme solution was used to investigate the effect of temperature, pH, metal ions and surfactants on the activity and stability of TESH (Wang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e values of the enzymes were determined by means of Michaelis-Menten plots at increasing \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate concentrations ranging from 5 to 170 mM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIsotope label\u003c/h2\u003e \u003cp\u003eThe synthesis of TA requires the participation of water molecules, we use H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO to label the reaction system. For single turnover reaction, 200 nmol of TESH and 40 nmol disodium \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate were dissolved in 500 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO, then incubated the mixture at 30\u0026deg;C for 12 h. For multiple turnover experiment, 4 nmol of TESH and 400 nmol disodium \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate were mixed in 500 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO and incubated at 30\u0026deg;C for 12 h. The reaction is terminated with an equal volume of methanol, then measured the molecular mass of the produced tartrate by G6465B Ultivo Triple Quadrupole LC/MS (Agilent, USA) (Bao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme assay\u003c/h2\u003e \u003cp\u003eTESH activity was assay at 37\u0026deg;C for 30 min in 1 mL of 0.2 M disodium \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate (pH 8.0). The quantity of \u003cem\u003emeso\u003c/em\u003e-TA was determined by Chirex 3126 (D)-penicillanmin (50\u0026times;4.6 mm) (Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). One unit of ESH activity was defined as the amount of the enzyme that generating 1\u0026micro;mol \u003cem\u003emeso\u003c/em\u003e-TA per minute. Specific activity was defined as the number of units per milligram protein. Protein was determined by the Bradford protein assay kit (Shanghai, China).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of TESH\u003c/h2\u003e \u003cp\u003eThe optimum temperature and pH of TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 were 45\u0026deg;C and pH 9.0, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However enzyme activity was almost lost when incubated at 45\u0026deg;C for 30 min, only 10% of residual activity remained when pH was lower than 5 or pH was higher than 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating poor temperature and pH stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003eStatistical analysis indicated that Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e were the activators of the enzyme, increased TESH activity significantly (P ＜ 0.001). Zn\u003csup\u003e2+\u003c/sup\u003eand Mn\u003csup\u003e2+\u003c/sup\u003e had strongly inhibited the activity of TESH ( P＜ 0.001). The addition of EDTA reduced the activity of the enzyme by 46.63%. SDS was a strong inhibitor, decreasing 95.85% activity of the enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The kinetic parameters were determined by Michaelis-Menten plotting method (GraphPad Prism 9.5). The vales of \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e were calculated to be 3.50 mM and 94.75 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig. S1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSequence comparison of TESH and reported EHs\u003c/h2\u003e \u003cp\u003eThe sequence alignment results indicated that TESH shows no homology to other reported ESHs (ESH(\u003cem\u003eL\u003c/em\u003e) belongs to HAD like superfamily, ESH(\u003cem\u003eD\u003c/em\u003e) belongs to BKACE superfamily) that belongs to MhpC superfamily. We found several structurally characterized proteins with low but considerable sequence identity (between 19% and 46%) were shown in a Clustal Omega (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Several residues were highly conserved: nine nonpolar amino acids (G23, G25, G33, G61, G63, L48, L59, A49, P235), two arginine (D58 and D104), two histidine (H103 and H272) and one lysine (Y211), polar amino acids play an important role in enzyme catalysis. Besides, E35 and H104 form ion pairs in StEH1, so we also designed corresponding Q36 mutated amino acid in TESH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHomology modeling of TESH\u003c/h2\u003e \u003cp\u003eWe selected 4NVR as the template structure to generate the 3D structure of the TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 with Swiss-Model, this template possesses the greatest amino acid sequence identity with TESH (46.52%). The root mean square deviation value of the modeled TESH was 0.068 \u0026Aring; (Fig. S2). The Ramachandran plot (Fig. S3) showed that the percentages of residues falling in disallowed regions, generously allowed regions, favorable regions and core regions were 0, 1.2, 9.3, and 89.5%, respectively. An overall plot showed that more than 95% of the residues were within the favorable region, which revealed that the quality of the model was good.\u003c/p\u003e \u003cp\u003eThe modeled 3D structure of TESH has the typical eight parallel β-sheets (residues 6\u0026ndash;10, 16\u0026ndash;22, 27\u0026ndash;31, 53\u0026ndash;57, 98\u0026ndash;103, 121\u0026ndash;127, 236\u0026ndash;241 and 263\u0026ndash;268) surrounded by α-helices and is covered by a cap-domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). It is similar with the structure from MhpC superfamily (StEH1, AD1, CorEH, and FAcD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate substrate was docked in the core of the modeled TESH structure, results showed that there were thirteen amino acids (H34, D104, R105, R108, D128, I129, Y147, H149, W150, V175, R179, Y211, H272) near the binding pocket, which were labeled on the modeled structure by PyMOL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSite-directed mutagenesis\u003c/h2\u003e \u003cp\u003eCombing the results of multiple sequence alignment and molecular docking, we selected fourteen amino acid sites to replaced their polar amino and conserved charged acid residues with another residues as follows: H and D by N; Q by E; R by K; I, V and W by A; Y by F. Purified protein were showed in Fig. S4. The results of site-directed mutagenesis revealed that six mutants showed relative activities that were only 2% or less of the wild-type enzyme\u0026rsquo;s activity: R108K, D128N, Y147F, H149N, W150A and Y211F. Four mutants showed the lower \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value than the wild-type: H34N, D104N, R105K, Y211F (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results revealed that the ten residues (H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272) played important roles in the catalysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of wild-type and mutant TESH enzyme.\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelative activity(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e(s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e(mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWile-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH34N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ36E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD104N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e54.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR105K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR108K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD128N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI129A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY147F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH149N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW150A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV175A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR179K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY211F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH272N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eThe specific activity of the wild-type was 124.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34 \u0026micro;mol min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInsert Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSingle and multiple turnover reaction of wild-type TESH in H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO\u003c/h2\u003e \u003cp\u003eThe single and multiple turnover reactions of TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 in H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO showed that most of the tartrate was \u003csup\u003e16\u003c/sup\u003eO-labeled in the single turnover reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), while most of the tartrate was \u003csup\u003e18\u003c/sup\u003eO-labeled in the multiple turnover reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggested that the catalytic mechanism of the TESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 same as most α/β hydrolases that proceeded by a two-step catalytic reaction involving the forming of an enzyme-substrate ester intermediate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe ten important catalytic residues of the TESH we obtained were aligned with the dehalogenase from \u003cem\u003eR. palustris\u003c/em\u003e CGA009 (FAcD), epoxide hydrolase from \u003cem\u003eCorynebacterium\u003c/em\u003e sp. C12 (CorEH), epoxide hydrolase from \u003cem\u003eA. radiobacter\u003c/em\u003e (AD1), epoxide hydrolase from \u003cem\u003eS. tuberosum (\u003c/em\u003eStEH1) and CFTR inhibitory factor Cif from \u003cem\u003eP. aeruginosa\u003c/em\u003e (Cif). We found that these ten residues were similar to the active sites of the FAcD, CorEH, AD1, StEH1 and Cif (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which have a two-step catalytic mechanism characterized by an aspartate-histidine-aspartate catalytic triad (Nardini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Chan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bahl et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bauer et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Schuiten et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the TESH may consists of the catalytic nucleophile Asp104, the charge relay acid Asp128 and the histidine base His272 to forming an Asp104-His272-Asp128 catalytic triad, which is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003cp\u003eLike most of EHs, Tyr residue that near the substrate plays an important role in help opening the ring. Firstly, they have hydrogen bond with the epoxide oxygen, which can position the substrate in the active site for nucleophilic attack. Then, tyrosine provide a proton to the epoxide oxygen during the ring-opening step (Yamada et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). While epoxide hydrolases usually use Tyr-Tyr pair to assist ring-opening (Nardini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Loo et al. 2006; Bauer et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), some epoxide hydrolases use a His-Tyr pair instead (Chan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bahl et al. 2012; Schuiten et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We propose Tyr211 co-action with Tyr147-His149-Trp150 to open the ring of \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). When nucleophilic Asp104 attack oxirane ring in the opposite side forming enzyme-substrate intermediate, Tyr211 co-action Tyr147-His149-Trp150 to form an oxyanion hole that stabilizes the oxyanion of the alkyl-enzyme intermediate formed by ring opening (Amrein et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn FAcD Arg111-Arg114 pair are the binding sites for carboxylic acids (Chan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), in CorEH Trp100 is the halide-stabilizing residue (Schuiten et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Trp38 and Phe108 have hydrogen bonds with Asp107 thus stabilize the intermediate (Nardini et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Glu35-His104 ion pair in StEH1 can control solvent access to the active site (Amrein et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Same as AD1, CFTR inhibitory factor Cif also have hydrogen bond between Phe63 and Ile130 which can stabilize intermediate (Bahl et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). According to homologous molecular docking results, Arg105 and Arg108 from TESH have hydrogen bond with the carboxylate binding site of \u003cem\u003etrans\u003c/em\u003e-epoxysuccinate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). H34 have polar contacts with D104 and Y211 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We propose like StEH1, H34 in TESH also play the same role as E35 is the part of a hydrogen-bond network that connects the active site with the solvent interface (Amrein et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). More experiments are needed to confirm this conjecture.\u003c/p\u003e \u003cp\u003eBased on the results and the above analysis, we speculated that the TESH adopted a two-step catalytic mechanism via Asp104-His272-Asp128 catalytic triad (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The catalytic mechanism of ESH(\u003cem\u003emeso\u003c/em\u003e) is similar to that of ESH(\u003cem\u003eL\u003c/em\u003e) from \u003cem\u003eR.opacus\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e sp. BK-58, which are also proceeded through the two-step mechanism involving the formation of a covalent intermediate (Pan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). But it is different from the ESH(\u003cem\u003eD\u003c/em\u003e) from \u003cem\u003eBordetella\u003c/em\u003e sp. BK-52, which catalyzes by means of a Zn\u003csup\u003e2+\u003c/sup\u003e-dependent, one-step mechanism (Dong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInsert Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTESH from \u003cem\u003eP. koreensis\u003c/em\u003e BK-9 belonged to MhpC superfamily and had a typical α/β hydrolase folding structure. Enzymatic results showed the optimum conditions of the enzyme were 45\u0026deg;C and pH 9.0, enzyme activity was strongly inhibited by Zn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e and SDS. Michaelis-Menten constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e was 3.50 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the constant of catalytic activities was 99.75 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the enantiomeric purity was higher than 99.9%. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272 were important residues involved in catalysis. Isotope labeling study suggested the enzyme acted via Asp104-His272-Asp128 two-step catalytic mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u0026nbsp; WNB and HFP contributed to the study conception and design. Material preparation, data collection and analysis were performed by HXL, JFY and RLZ. The first draft of the manuscript was written by HXL and all authors commented on previous version of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp; \u0026nbsp;This study was funded by the Huzhou Scientific and Technological Project (2022GZ56) and the Education of Zhejiang Province of China (Y202248484).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval \u0026nbsp;\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent \u0026nbsp;\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all individual participants included in the study.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllen RH, Jakoby WB (1969) Tartaric acid metabolism. 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Molecules 24: 903-14. http://dx.doi.org/10.3390/molecules24050903\u003c/li\u003e\n\u003cli\u003eYamada T, Morisseau C, Maxwell JE, Argiriadi MA, Christianson DW, Hammock BD (2000) Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase. J Biol Chem 275: 23082-88. http://dx.doi.org/10.1074/jbc.M001464200\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"trans-epoxysuccinate hydrolase, Pseudomonas koreensis, meso-tartaric acid, catalytic mechanism","lastPublishedDoi":"10.21203/rs.3.rs-3437865/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3437865/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThis study aimed to discuss the essential amino acid residues and catalytic mechanism of \u003cem\u003etrans\u003c/em\u003e-epoxycussinate hydrolase from \u003cem\u003ePseudomonas koreensis\u003c/em\u003e for production of \u003cem\u003emeso\u003c/em\u003e-tartaric acid.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe optimum conditions of the enzyme were 45\u0026deg;C and pH 9.0, respectively. It was strongly inhibited by Zn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e and SDS. Michaelis-Menten enzyme kinetics analysis gave a \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of 3.50 mM and a \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e of 99.75 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the EE value was higher than 99.9%. Multiple sequence alignment and homology modeling showed that the enzyme belonged to MhpC superfamily and had a typical α/β hydrolase folding structure. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211 and H272 were important catalytic residues. \u003csup\u003e18\u003c/sup\u003eO-labeling study suggested the enzyme acted via two-step catalytic mechanism.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe structure and catalytic mechanism of \u003cem\u003etrans\u003c/em\u003e-epoxycussinate hydrolase were firstly reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad were proposed.\u003c/p\u003e","manuscriptTitle":"Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-30 18:58:56","doi":"10.21203/rs.3.rs-3437865/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-06T13:16:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-27T13:48:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-14T14:54:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2023-11-13T08:30:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2023-10-20T08:31:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ba7b1db6-47e8-4ec4-84ff-1d028ad75644","owner":[],"postedDate":"November 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-14T00:41:47+00:00","versionOfRecord":{"articleIdentity":"rs-3437865","link":"https://doi.org/10.1007/s10529-024-03490-3","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2024-05-13 00:41:47","publishedOnDateReadable":"May 13th, 2024"},"versionCreatedAt":"2023-11-30 18:58:56","video":"","vorDoi":"10.1007/s10529-024-03490-3","vorDoiUrl":"https://doi.org/10.1007/s10529-024-03490-3","workflowStages":[]},"version":"v1","identity":"rs-3437865","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3437865","identity":"rs-3437865","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}