Biotechnol Lett (2014) 36:2537–2544 DOI 10.1007/s10529-014-1638-7

ORIGINAL RESEARCH PAPER

Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp. Yongqing Cheng • Haifeng Pan • Wenna Bao • Weirong Sun • Zhipeng Xie • Jianguo Zhang • Yuhua Zhao

Received: 17 June 2014 / Accepted: 11 August 2014 / Published online: 13 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to L (?)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel bsheets flanked by seven a-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket

Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1638-7) contains supplementary material, which is available to authorized users. Y. Cheng  H. Pan  Z. Xie (&)  J. Zhang Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, Zhejiang, China e-mail: [email protected] H. Pan  W. Bao  W. Sun  Z. Xie  J. Zhang Hangzhou Bioking Biochemical Engineering Co., Ltd, Hangzhou 311106, Zhejiang, China Y. Zhao Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, Zhejiang, China

between the two domains. An 18O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism. Keywords Catalytic mechanism  cis-epoxysuccinate hydrolase  Epoxide hydrolase  Homology modeling  Klebsiella sp  L(?)-Tartaric acid

Introduction Epoxide hydrolases (EHs; EC 3.3.2.3) are a group of functionally-related enzymes that can catalyze the enantioselective hydrolysis of epoxide to the corresponding product (Steinreiber and Faber 2001). Although EHs are found in a variety of sources, those from microorganisms have gained attention owing to their efficient catalysis for the production of fine organic chemicals (de Vries and Janssen 2003). Cisepoxysuccinate hydrolase (ESH) catalyzes the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to the corresponding tartaric acid or tartrate (Willaert and de Vuyst 2006). L(?)-Tartaric acid is widely used in the food, building, pharmaceutical, and cement industries (Bucˇko et al. 2005). Traditionally, L(?)tartaric acid, which is abundant in grapes, is extracted from winery waste. However, production of L(?)tartaric acid is strongly influenced by the yield and quality of grapes, which are dependent on the climate.

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Currently, the microbial method is considered to be more suitable for the production of L(?)-tartaric acid. Cis-epoxysuccinic acid has been hydrolyzed by ESHs from various microbial species and several ESHs have been purified and characterized. Only two genes encoding ESH for L(?)-tartaric acid production have been cloned: from Rhodococcus opacus (Liu et al. 2007) and from Nocardia tartaricans (Wang et al. 2012). The nucleotide sequences of both genes, however, are identical (Liu et al. 2007; Wang et al. 2012). The possible structure and catalytic mechanism of the ESH from R. opacus have been predicted (Pan et al. 2011). We purified a novel ESH that hydrolyzed cis-epoxysuccinate to L(?)-tartaric acid more efficiently with better thermal and pH stabilities from Klebsiella sp. BK-58 in our previous work (Cheng et al. doi:10.1007/s10529-014-1614-2). Production conditions of L(?)-tartaric acid could be widened due to its good thermal and pH stabilities. It also had a stronger affinity to cis-epoxysuccinate and a higher catalytic efficiency, which revealed that the ESH from Klebsiella sp. BK-58 could be of significant importance in the industrial production of L(?)-tartaric acid. Therefore, it is necessary to clone the ESH gene from Klebsiella sp. BK-58 and to construct the genetic engineering bacteria for the industrial production of L(?)-tartaric acid. It is also of significant importance to investigate the structure and catalytic mechanism of the enzyme. In this study, we have cloned the ESH gene from Klebsiella sp. BK-58 and expressed it in Escherichia coli. Multiple alignment, homology modeling, site-directed mutagenesis, circular dichroism spectra analysis and 18Olabeling studies were carried out to determine the structure and reaction mechanism of the novel ESH.

Materials and methods Strains, plasmids and reagents Klebsiella sp. BK-58 was isolated and stored in our laboratory. E. coli DH5a was used as host for DNA manipulations and E. coli BL21 (DE3) was used for gene expression. Primers summarized in Supplementary Table 1 were synthesized by Sangon Biotech (Shanghai, China). BamHI, NdeI, T4 DNA ligase, pUCm-T, pET-22b, bovine serum albumin and IPTG were all obtained from Sangon Biotech. H18 2 O (purity: 97 %) was obtained from Shanghai Research Institute

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of Chemical Industry (Shanghai, China). All other chemicals used in this study were of molecular biology grade. Protein terminal sequencing In order to clone the ESH gene from Klebsiella sp. BK-58, we first got the terminal amino acid sequences of the protein. The ESH from Klebsiella sp. BK-58 was purified to homogeneity in our previous work (Cheng et al. 2014). The purified enzyme was electroblotted onto a PVDF membrane. The transferred protein band was subjected to N-terminal and Cterminal sequence analysis on an ABI Procise N sequencer and an ABI Procise C sequencer (Applied Biosystems, CA, USA). Cloning and expression of ESH The genomic DNA of Klebsiella sp. BK-58 was extracted with the AxyPrep Bacterial Genomic DNA MiniPrep Kit (Axygen). Based on the results of N-terminal and Cterminal amino acid sequence analysis, primers P1 and P2 (Supplementary Table 1) were designed. The ESH gene was amplified with the two primers and the PCR products were purified using a DNA Gel Extraction Kit (Axygen). The purified PCR fragment was ligated with pUCm-T by T4 DNA ligase, and then the recombinant plasmid was transferred into E. coli DH5a competent cells. Positive clones were sequenced. Based on the sequencing results, primers P3 and P4 (Supplementary Table 1), with the added compatible restriction sites of BamHI and NdeI respectively, were designed to amplify the ESH gene. The PCR products and the plasmid pET-22b were digested with BamHI and NdeI, ligated by T4 DNA ligase, and then the recombinant plasmid pET-22b-ESH was transferred into E. coli BL21 (DE3) competent cells. A positive clone was selected and inoculated into LB medium with 0.01 % (w/v) ampicillin. The culture solution was incubated at 37 °C for 2 h, and then IPTG was added at 0.1 mM to initiate over-expression of ESH for 12 h at 37 °C. Multiple sequence alignment and site-directed mutagenesis We searched the Protein Data Bank (PDB) database with the ESH sequence of Klebsiella sp. BK-58 as the

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probe and aligned the sequences with ClustalW2 (Larkin et al. 2007). Based on the reaction mechanism study of the ESH from R. opacus by Pan et al. (2011) and the results of the alignment, we carried out sitedirected mutagenesis and ESH mutants (D48 N, T52A, R85 K, N165D, K195R, Y201F, A219S, H221 N, and D224 N) were produced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) with two reverse complement primers; the sequences were confirmed by DNA sequencing. The mutant enzymes and nine forward primers are shown in Supplementary Table 1.

Enzyme purification and characterization Both wild-type and mutant ESHs were expressed in E. coli BL21 (DE3) cells, which were cultivated at 25 °C for 12 h in LB medium containing 0.01 % (w/v) ampicillin and 0.1 mM IPTG. After centrifugation, the harvested cells were disrupted by ultrasonication. After centrifugation, the supernatant was applied to a His-binding resin column (ZHbio, Hangzhou, China). The fractions containing ESH were pooled, ultra filtrated, and identified by SDSPAGE. ESH activity was determined by the determination of the amount of L(?)-tartaric acid from cis-epoxysuccinate. The reaction mixture in 1 ml contained 20 ll enzyme solution, 0.1 M phosphate buffer (pH 8) and 0.2 M disodium cis-epoxysuccinate. The reactions were carried out at 37 °C for 20 min and were stopped by adding 400 ll 1 M sulfuric acid to the mixture. One unit (1 U) of ESH activity was defined as the amount of enzyme capable of generating 1 lmol L(?)-tartaric acid per minute at pH 8 and 37 °C. Specific activity was defined as the number of units per mg protein. Protein was quantitatively determined by the Bradford method with bovine serum albumin as the standard. Kinetic parameters of nine mutant ESHs were determined by assaying purified enzyme at increasing substrate concentrations ranging from 10 to 100 mM for triplicate determinations. Km, Vmax and kcat were determined using Lineweaver–Burk plots. Circular dichroism spectra of wild type and mutant ESHs were measured on a Jasco-815 spectropolarimeter and were expressed as the mean residue ellipticities (h deg cm2 dmol-1).

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Homology modeling A 3D structure of the ESH from Klebsiella sp. BK-58 was generated with the MODELLER program (Version 9.12) using 3UMC, which possesses the greatest amino acid sequence identity with ESH (36 %) in the PDB database, as the template structure. The molecular graphics figures were generated with PyMol (Version 1.5). Nucleotide sequence accession number The nucleotide sequence of ESH from Klebsiella sp. BK-58 was submitted to the GenBank database under accession number KF977193.

Results and discussion Protein terminal sequencing In order to clone the ESH gene from Klebsiella sp. BK-58, the terminal amino acids of the purified protein was sequenced. N-terminal and C-terminal sequence analysis determined ten amino acids at two ends of the ESH from Klebsiella sp. BK-58 respectively. The N-terminal amino acid sequence was MKFSGASLFA, and the C-terminal was VVELAGMLGA. Cloning and expression of ESH Based on the result of the N-terminal and C-terminal sequence analysis, primers P1 and P2 (Supplementary Table 1) were designed to amplify the ESH-encoding gene, and a PCR fragment of approx. 0.8 kb (Fig. 1a) was amplified from Klebsiella sp. BK-58 and sequenced. The sequence analysis using DNAStar indicated that the fragment contained an ORF of 825 bp encoding a mature polypeptide of 274 amino acid residues. The deduced molecular mass and pI of ESH from Klebsiella sp. BK-58 was 30.1 kDa and 8.06, respectively. The ORF region was expressed in E. coli BL21 (DE3) and the recombinant E. coli demonstrated high-level expression of recombinant protein (Fig. 1b). The recombinant protein was extracted from the soluble fraction of the bacterial crude and purified by immobilized metal affinity chromatography. The band representing ESH was

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Characterization of the mutant enzymes

Fig. 1 a Agarose gel electrophoresis of the ESH gene cloned from Klebsiella sp. BK-58. Lane 1, Marker; Lane 2, PCR products with P1 and P2. b Identification of ESH expression by SDS-PAGE. Lane 1, Marker; Lane 2, Total protein expressed by E. coli with recombinant pET-22b-ESH induced with 0.1 mM IPTG

between 25 and 35 kDa based on SDS-PAGE analysis (Supplementary Fig. 1a), which was consistent with the theoretical molecular weight of the mature protein (30.1 kDa). Sequence comparison of ESH and reported EHs The amino acid sequence of ESH from Klebsiella sp. BK-58 was compared with PDB database using ClustalW2 program. The sequence alignment results showed that it belonged to the haloacid dehalogenase (HAD)-like superfamily, but it showed only 36 % identity with the sequence of ESHs from Rhodococcus opacus and Nocardia tartaricans (Liu et al. 2007; Wang et al. 2012). ESHs from R. opacus and N. tartaricans, and a selection of proteins (Schmidberger et al. 2007; Ridder et al. 1999; Novak et al. 2013; Hisano et al. 1996; Li et al. 1998) with low but significant sequence identity (between 27 and 36 %), are shown in a ClustalW2 alignment result (Fig. 2). The secondary structure of ESH was predicted by PredictProtein server (Rost et al. 2004) and it was similar to the secondary structures of proteins from the amino acid sequence alignment (Fig. 2).

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Although the amino acids sequence of the ESH from Klebsiella sp. BK-58 showed only 36 % identity with that of the ESH from R. opacus, the results of multiple sequence alignment (Fig. 2) suggested that the two ESHs had similar conservative positions. The nine highly conserved residues (D18, T22, R55, N134, K164, Y170, A188, H190, and D193) of ESH from R. opacus played important roles in the catalysis (Pan et al. 2011). The residues (D48, T52, R85, N165, K195, Y201, A219, H221, and D224) of ESH from Klebsiella sp. BK-58 were also highly conserved and lay on sites topologically equivalent to those of the catalysis involved residues of ESH from R. opacus (Fig. 2). Therefore, we carried out site-directed mutagenesis and ESH mutants (D48 N, T52A, R85 K, N165D, K195R, Y201F, A219S, H221 N, and D224 N) were produced to research whether the ESH from Klebsiella sp. BK-58 had similar catalytic residues. The enzymes were purified by immobilized metal affinity chromatography. The mutant enzymes were homogeneous upon SDS-PAGE analysis (Supplementary Fig. 1b) and migrated at the same size compared with the wildtype enzyme (Supplementary Fig. 1a). The nine mutants all showed no more than 10 % relative activities compared with the wild-type enzyme (Table 1). The Km values of the nine mutants were slightly larger than the wild-type enzyme, but their kcat values were significantly lower than the wild-type enzyme, revealing that residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 are likely important residues involved in catalysis. Circular dichroism spectra of these nine mutants were not significantly different from the spectrum of the wildtype enzyme (Table 2). Homology modeling of ESH 3UMC, which possesses the greatest amino acid sequence identity with ESH (36 %), was used as the template structure to generate the 3D structure of the ESH from Klebsiella sp. BK-58 with the MODELLER program. The result of the modeling was analyzed and verified by a Ramachandran plot (Supplementary Fig. 2), which showed that the percentages of residues falling in disallowed regions, generously allowed regions, favorable allowed regions, and core regions were 0, 1, 8.7, and 90.4 %, respectively. An overall

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Fig. 2 Sequence alignments for ESH from Klebsiella sp. BK58, ESH from R. opacus (ESH*), and seven structurally characterized proteins from the PDB database. The sequences were aligned with ClustalW2 and were shown in order of decreasing sequence identity compared to ESH. Identical amino acids are marked with an asterisk, conserved substitution residues with a colon, semiconserved substitution residues with a period, and residues mutagenized in our study with a number sign . a-Helices are shaded black,, and b-sheets are shaded gray. Sequences: 3UMC, haloacid dehalogenase from Pseudomonas

aeruginosa PAO1 (NP_249501); 3UMG, haloacid dehalogenase from Rhodococcus jostii RHA1 (YP_700224); 2NO4, 2-haloalkanoic acid dehalogenase IVA from Burkholderia cepacia MBA4 (Q51645); 1QQ5, L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 (Q60099); 2YML, Lhaloacid dehalogenase from Rhodobacteraceae (PDB ID: 2YML); 3UM9, haloacid dehalogenase from Polaromonas sp. JS666 (YP_547390); 1JUD, 2-haloacid dehalogenase from Pseudomonas sp. YL (Q53464)

plot showed that the favorable region possessed more than 95 % of the residues, which revealed that the quality of the model was good. The overall structure of the ESH consisted of two structurally distinct domains (Fig. 3): a core domain that had an a/b structure formed by six-stranded parallel b-sheets flanked by

five long a-helices and two short a-helices, and a subdomain that had a four helix bundle structure. This structure was different than the classic a/b-fold including eight-stranded parallel b-sheets flanked by a-helices (Ollis et al. 1992). However, it was similar to the modeled ESH structure from R. opacus and other

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Table 1 Characterization of wild-type and mutant ESHs from Klebsiella sp. BK-58 Relative activity (%)a

Enzyme

Km (mM)

kcat (s-1)

kcat/Km (mM-1s-1)

Wild-typeb

100 ± 0.7

19.3 ± 1.8

220 ± 8.9

11.4 ± 0.7

D48 N

1.3 ± 0.05

29.2 ± 2.2

1.8 ± 0.3

0.06 ± 0.01

T52A R85 K

Not detectedc 1 ± 0.08

29.6 ± 1.1

3.3 ± 0.7

0.11 ± 0.02

N165D

4.6 ± 0.6

27.9 ± 1.4

21 ± 1.1

0.75 ± 0.08

K195R

0.4 ± 0.03

26.7 ± 0.8

2.9 ± 0.2

Y201F

0.4 ± 0.02

19.5 ± 1.2

1 ± 0.1

0.05 ± 0.009

A219S

0.4 ± 0.08

20.1 ± 0.5

0.9 ± 0.1

0.05 ± 0.006

H221 N

0.7 ± 0.07

40.4 ± 2.3

1.6 ± 0.3

0.04 ± 0.009

D224 N

Not detected

a

0.11 ± 0.01

The specific activity of the wild-type ESH was 47 lmol min-1 mg-1

b

Characterization of wild-type ESH was from our previous work (Supplementary Information for review only, Cheng et al. unpublished data) c

The activity of a mutant enzyme could not be determined if its specific activity was lower than 0.1 lmol min-1 mg-1

Table 2 Predicted secondary structures of wild-type and mutant ESHs from Klebsiella sp. BK-58 Enzyme

Secondary structure contents (%) Helix

Sheet

Wild-type D48 N

34.2 29.4

15.4 24.5

T52A

29.6

23.1

R85 K

29.8

21.3

N165D

28.4

20.0

K195R

28.5

16.7

Y201F

28.2

16.1

A219S

30.8

17.0

H221 N

28.7

19.6

D224 N

27.1

18.1

three members (DehIVa, DhlB, and L -DEX) of the HAD-like superfamily with reported crystal structures (Pan et al. 2011; Schmidberger et al. 2007; Ridder et al. 1999; Hisano et al. 1996; Li et al. 1998). They all have a mixed a/b core domain and a four-helix bundle domain. These findings suggested that our enzyme had a typical two-domain structure of the HAD-like superfamily, just as the ESH from R. opacus. The modeling result also corroborated the secondary structure predication by the PredictProtein server (Fig. 2). The nine important catalytic residues (D48, T52, R85, N165, K195, Y201, A219, H221, and D224) were labeled on the modeled structure of ESH (Fig. 3) by PyMOL.

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Fig. 3 Modeled homology structure of the ESH from Klebsiella sp. BK-58. The spirals represent a-helices, and the arrows represent b-sheets. Nine residues involved in the catalysis reaction are shown

Reaction mechanism of the ESH from Klebsiella sp. BK-58 The single and multiple turnover reactions of ESH from R. opacus in H18 2 O (Pan et al. 2011) showed that most of the tartrate was 16O-labeled in the multiple turnover reaction, while most of the tartrate was 18O-labeled in the single turnover reaction. The results of the ESH from Klebsiella sp. BK-58 were similar and are shown in Fig. 4. Under single turnover conditions, more than 95 % of the tartrate was labeled with 16O (Fig. 4b), while more than 95 % of the tartrate was labeled with 18 O (Fig. 4c) under multiple turnover conditions. These

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Fig. 4 Ion spray mass spectra of tartrate produced by the ESH from Klebsiella sp. BK-58 in H18 2 O. a control station; b single turnover reaction; c, multiple turnover reaction. For b, 800 nmol wild-type ESH, 40 nmol disodium cis-epoxysuccinate and 200 ll 10 mM previously lyophilized Tris–HCl (pH 7) were mixed in 200 ll H18 2 O and incubated at 30 °C for 12 h. For c 20 nmol wild-type ESH, 4 lmol disodium cis-epoxysuccinate and 200 ll 10 mM previously lyophilized Tris–HCl (pH 7) were mixed in 200 ll H18 2 O and incubated at 30 °C for 12 h. The reaction mixtures were analyzed by LCQ Deca XP mass spectrometer 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 4 kV, and the orifice voltage was optimized at 50 V

results suggested that the catalytic mechanism of the ESH from Klebsiella sp. BK-58 proceeded by a two-step catalytic reaction involving the formation of an enzymesubstrate ester intermediate. The results of site-directed mutagenesis (Table 1) revealed that the nine residues (D48, T52, R85, N165, K195, Y201, A219, H221, and D224) played important roles in the catalysis. These nine functionally important residues lay on sites topologically equivalent to those of the active site residues (D18, T22, R55, N134, K164, Y170, A188, H190, and D193) of ESH from R. opacus (Pan et al. 2011), which suggested that the two enzymes from different strains might have the same reaction mechanism. The result of 18O-labeling reaction (Fig. 4) showed that the ESH from Klebsiella sp. BK-58 had a two-step catalytic mechanism, which was the same with the ESH from R. opacus and four members (DehIVa, DhlB, L-DEX, and DehRhb) of the HAD-like superfamily with reported crystal structures and reaction mechanisms (Schmidberger et al. 2007;

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Fig. 5 Proposed reaction mechanism of ESH from Klebsiella sp. BK-58. First, the carboxylate group of Asp48 nucleophilically attacks the carbon atoms in the oxirane ring of the substrate to form an enzyme-substrate intermediate jointed by an ester bond; then, the ester bond is hydrolyzed by an active water molecule activated by His221, which makes an ion pair with Asp224 leading to the formation of L(?)-tartaric acid and the release of the free halide

Ridder et al. 1999; Novak et al. 2013; Hisano et al. 1996; Li et al. 1998). The active site residues of ESH from R. opacus had been proved to lie on sites topologically equivalent to the active site residues of DehIVa, DhlB and L-DEX by Pan et al. (2011). Therefore our enzyme from Klebsiella sp. BK-58 is in the same situation. The DehIVa, DhlB and L-DEX have a two-step catalytic mechanism characterized by an Asp-Asn-Asp catalytic triad (Schmidberger et al. 2007; Ridder et al. 1999; Hisano et al. 1996; Li et al. 1998). According to the result of sequence alignment (Fig. 2) and 18O-labeling reaction, and the analysis above, we can predict that the ESH from Klebsiella sp. BK-58 has a two-step catalytic mechanism achieved by an Asp48-His221-Asp224 catalytic triad where histamine replaces the asparagine. The detailed procedure is shown in Fig. 5. This catalytic mechanism of our ESH is similar with that of the ESH from R. opacus with an Asp48-His221-Asp224 catalytic triad (Pan

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et al. 2011). Different from DehIVa, DhlB and L-DEX, the DehRhb contains a novel His/Glu dyad that could activate the catalytic water (Novak et al. 2013). The replacement of asparagine by histimine of the DehRhb is the same with our ESH. Overall, we can predict that the ESH from Klebsiella sp. BK-58 has a two-step catalytic mechanism achieved by an Asp48His221-Asp224 catalytic triad (Fig. 5), which is similar with that of the ESH from R. opacus.

Conclusions The gene encoding a novel ESH, which hydrolyzes cisepoxysuccinate to L(?)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in E. coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. The enzyme belonged to the HAD-like superfamily, had a twodomain 3D structure and catalyzed epoxide hydrolysis by a two-step mechanism, which was similar to that of the ESH from R. opacus. D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were indispensable for catalytic functions and further crystallographic study of the enzyme is now being carried out to determine the precise roles of these catalytic residues and the catalytic mechanism in more detail. Acknowledgments This project is supported by the High Technology Research and Development Program of China (863 Program) (2014AA022105; 2012AA06A203), the National Natural Science Foundation of China (31300661, 31070079; 41271335), the National Key Technology Rand D Program (2012BAC17B04), the Science and Technology Project of Zhejiang Province (2011C13016; 2013C3303), and the Environmental Science Project of Zhejiang Province (2012B006). Supporting information Supplementary Table 1—Primers used in our study Supplementary Figure 1—SDS-PAGE of purified ESH from Klebsiella sp. BK-58 Supplementary Figure 2—Ramachandran plot of the modeled 3D structure of ESH from Klebsiella sp. BK-58

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Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to L (+)-tartaric acid, was cloned from Klebsiella sp. BK-...
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