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FEBS Letters 585 (2011) 2545–2550

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Site-directed mutagenesis of epoxide to probe catalytic amino acid residues and reaction mechanism ⇑ Haifeng Pan a,1, Zhipeng Xie a,1, Wenna Bao a, Yongqing Cheng a, Jianguo Zhang a,b, , Yongquan Li a

a Institute of Biochemistry, Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China b Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou 311106, China

article info abstract

Article history: Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuc- Received 12 May 2011 cinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloac- Revised 4 July 2011 etate dehalogenase (16–23% identity). To identify catalytically important residues, we mutated 29 Accepted 4 July 2011 highly conserved charged and polar amino acid residues (except for one alanine). The replacement Available online 13 July 2011 of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the activ- Edited by Richard Cogdell ity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction Keywords: Epoxide hydrolase mechanism. Site-directed mutagenesis Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Mechanism Rhodococcus opacus

1. Introduction coccus [6], can catalyze stereospecific hydrolysis of cis- epoxysuccinate to L(+)-tartrate requiring neither cofactors, pros- Epoxide (EHs, EC 3.3.2.3), ubiquitous in nature, cat- thetic groups nor metal ions for their activities [1]. Only the pri- alyze the hydrolysis of epoxides to their corresponding diols. Many mary structure of Rhodococcus opacus EH (GenBank ID: studies focus on mammalian EHs. In recent years, bacterial EHs DQ471957) has been determined [6]. However, the relationship have been recognized as versatile biocatalysts for preparation of between its primary structure and function has never been re- enantiopure pharmaceuticals and other fine chemicals owing to ported, which is very interesting not only from the view point of the advantages of high stereospecificity and availability [1]. The basic enzymology, but also from the point of view of the molecular industrial synthesis of L(+)-tartaric acid is the first application of design showing more efficient catalysis. an EH catalyzed meso-epoxide hydrolysis [2]. The aim of this study is to identify catalytically important resi- L(+)-Tartaric acid is widely used in the food industry, pharma- dues and propose its reaction mechanism through sequence simi- ceutical industry, chemical analysis, textile industry and building larity analysis, site-directed mutagenesis, characterization of industry. Various bacterial EHs from different species, such as Rhi- mutant and single and multiple turnover enzyme reac- 18 zobium, Pseudomonas [3], Nocardia [4], Corynebacterium [5], Rhodo- tions in H2 O. The results showed that D18, D193, R55, K164, H190, T22, Y170, N134 and A188 were involved in the catalysis and the enzyme reaction proceeded through a two-step mecha- Abbreviations: EH, epoxide hydrolase; HAD, haloacid dehalogenase; EE, enan- nism. The role of these residues is discussed and a new two-step tiomeric excess; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electro- mechanism proposed. phoresis; Hdl, L-2-haloacid dehalogenase IVa from Pseudomonas cepacia MBA4; DehCI, L-2-haloacid dehalogenase I from Pseudomonas sp. strain CBS3; DehH2, haloacetate dehalogenase H-2 from Moraxella sp. strain B; DhlS5I, L-2-haloacid 2. Materials and methods dehalogenase from Agrobacterium tumefaciens RS5; L-DEX, L-2-haloacid dehalogen- ase from Pseudomonas sp. strain YL; DhlB, L-2-haloacid dehalogenase from 2.1. Construction of recombinant Escherichia coli for expression of EH Xanthobacter autotrophicus GJ10 ⇑ Corresponding author at: Institute of Biochemistry, Zhejiang University, Yuhangtang Road 388, Hangzhou 310058, China. Fax: +86 571 88206983. Rhodococcus opacus was cultivated [6] and its genomic DNA was E-mail addresses: [email protected], [email protected] (J. Zhang). extracted using EZ-10 spin column genomic DNA isolation kit (Bio 1 The authors wish it to be known that, in their opinion, the first two authors Basic Inc). The EH from Rhodococcus opacus was amplified by should be regarded as joint first authors.

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.07.006 2546 H. Pan et al. / FEBS Letters 585 (2011) 2545–2550

PCR using the following primers: forward primer, 50-CCATGGG containing EH were pooled, concentrated by ultrafiltration, stored CCATCACCATCACCATCACATGCAACTGAACAATGCG-30 (start codon in 10 mM Tris-HCl (pH 7.0) at 4 °C and identified by sodium dodecyl shown in bold; NcoI site underlined; (His)6-tag italics); reverse pri- sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). mer, 50-GGATCCTCAATCGATACCGGCAGTTC-30 (stop codon shown in bold; BamHI site underlined). Then the amplified EH fragment 2.4. Enzyme and protein assay was cloned into pTrc99A to be pTrc99A-EH and transformed into Escherichia coli JM109. EH activity was assayed at 30 °C for 20 min in 1 ml 80 mM diso- dium cis-epoxysuccinate (pH 7.0). The quantity of tartaric acid was 2.2. Site-directed mutagenesis measured according to our previous work [7]. One unit of enzyme was defined as the amount of the enzyme that generated 1 lmol of The site-directed mutagenesis was carried out using Quik- tartaric acid per minute and the number of such units per mg of ChangeÒ Lightning Site-Directed Mutagenesis Kits (Stratagene) protein was defined as specific activity. Protein assaying was per- with two reverse complement primers and the substitutions were formed with a Bio-Rad protein assay kit. confirmed by DNA sequencing. The mutant enzymes and synthetic mutagenic forward primers were as follows (the underlines indi- 2.5. Kinetic studies cate the mutagenized nucleotides):

Km and Kcat of wild-type and mutant EHs were determined by D18 N: 50-TCCGGGCCCTGCTTTTCAACGTGCAGGGGACTCTG-30 Lineweaver-Burk plot at increasing concentration rang- D25 N: 50-CAGGGGACTCTGACAAATTTCCGTTCCACACTC-30 ing from 10 mM to 160 mM for triplicate determinations. D60 N: 50-CGCGGCTGCTATCGAAACGAGCTCGATTCCTTGGT-30 D80 N: 50-GCGCCGTGTACCGAAATTCTCTTATCAATCTTCTCG-30 2.6. Enantiomeric excess assay D115 N: 50-CTTCGGTCGTGGCCGAACGTCCCCTCTGGATTGGA-30 D152 N: 50-GCCAAACTGCAATGGAACGCTGTTCTTTCAGC-30 Transformation was performed under standard reaction and the D193 N: 50-TCGCCTCCCATGCATACAATCTCGAAGCGGCGC-30 enantioselectivity of was evaluated with enantiomeric ex- R55 K: 50-AGGAATTGGTCGACCAATGGAAGGGCTGCTATCGA- cess (EE) value determined by HPLC (Agilent 1200) according to GACGA-30 our previous work [8]. R209 K: 50-CACAGCGTACGTCAGAAAGCCACTGGAATACGG-30 K70R: 50-CTTGGTCAAACAGGAGAGATGGCGCTCGGTCC-30 2.7. Circular dichroism analysis K164R: 50-CTCAACTCTTTGGAGCCTACAGGCCCCACCGGTCAAC-30 H190 N: 50-TCCTCATGGTCGCCTCCAATGCATACGATCTC- CD spectra were measured on a Jasco-815 spectropolarimeter GAAGCG-30 over the range of 250-190 nm. Three spectra were accumulated S28A: 50-CTCTGACAGATTTCCGTGCCACACTCATCGAGC-30 using the following conditions: protein concentration, 0.112 mg/ S81A: 50-GCCGTGTACCGAGATGCTCTTATCAATCTTCTCGC-30 ml; buffer, 10 mM Tris-HCl (pH 7.0); sensitivity, 2 mdeg/cm; scan- S156A: 50-TGGGACGCTGTTCTTGCAGCTCAACTCTTTGG-30 ning speed, 50 nm/min; band width, 1 nm; path length, 0.1 cm at S189A: 50-ATCCTCATGGTCGCCGCCCATGCATACGATCT-30 25 °C. CD data were expressed as mean residue ellipticities, [h], T22A: 50-TTTTCGACGTGCAGGGGGCTCTGACAGATTTCCGTTCC- in degcm2dmol-1, and the content of secondary structural ele- 30 ments was predicted using K2D program. T133A: 50-CTCGTCGCGGCACTGGCGAATGCGGACTTTTCTGCC-30 0 0 18 Y170F: 5 -GCCCCACCGGTCAACATTTGAGGGAGCCGCGACA-3 2.8. Single and multiple turnover reactions of wild-type EH in H2 O Y163F: 50-CAGCTCAACTCTTTGGAGCCTTCAAGCCCCACCGGTC-30 Y192F: 50-CGCCTCCCATGCATTCGATCTCGAAGCGG-30 For a typical single turnover experiment, 800 nmol of the wild- W54F: 50-AGGAATTGGTCGACCAATTCCGCGGCTGCTATCG-30 type EH in 200 ll of 10 mM Tris-HCl buffer (pH 7.0) was lyophi- W71F: 50-CTTGGTCAAACAGGAGAAATTTCGCTCGGTCCGCG-30 lized. The reaction was initiated by dissolving the dried enzyme 0 0 18 W107F: 5 -ATTGCTGACCGATGGTTTTGAACGTCTTCGGTCG-3 in 200 llofH2 O (purity: 97%, ShangHai Research Institute of W113F: 50-GAACGTCTTCGGTCGTTTCCGGACGTCCCCT-30 Chemical Industry, Shanghai, China) containing 40 nmol disodium M186L: 50-CCGTCAGAGATCCTCCTGGTCGCCTCCCAT-30 cis-epoxysuccinate, and the mixture was incubated at 30 °C for N134D: 50-CGCGGCACTGACGGATGCGGACTTTTCTGC-30 12 h. For a multiple turnover experiment, 20 nmol of EH, 4 lmol Q158E: 50-GACGCTGTTCTTTCAGCTGAACTCTTTGGAGCCTACAA- of disodium cis-epoxysuccinate and 2 lmol of Tris-HCl (pH 7.0) 0 18 3 previously lyophilized were mixed in 200 llofH2 O and incu- A188S: 50-GTCAGAGATCCTCATGGTCTCCTCCCATGCATAC- bated at 30 °C for 12 h. The reaction mixtures were ultrafiltrated, GATCT-30 diluted with equal volume of methanol, and then introduced into the mass spectrometer. The molecular mass of the produced tar- 2.3. Cultivation of the cells and purification of the enzymes trate was measured by LCQ Deca XP mass spectrometer system equipped with an ionspray ion source in the negative ion mode Both wild-type and mutant EHs were expressed in E. coli JM109. (Thermo Finnigan, USA). E. coli cells were cultivated at 25 °C for 24 h in Luria-Bertani medium (1% peptone, 0.5% yeast extract and 1% NaCl, pH 7.0) containing 100 lg/ml ampicillin and 0.2 mM isopropyl b-D-1-thiogalactopyra- 3. Results noside. The cells were centrifuged, washed and suspended in equil- ibration buffer (50 mM Tris-HCl, 0.5 M NaCl, pH 7.0). Cells were 3.1. Sequence comparison with EH broken by sonication with ice cooling and the cell debris was removed by centrifugation. The supernatant solution was applied The amino acid sequence of EH from Rhodococcus opacus was on a His binding resin (York Biotech, Shanghai, China), the non- compared with SWISS-PROT protein database using BLAST pro- absorbed protein fraction were eluted with 20 mM imidazole in gram. The results showed that it belongs to the haloacid dehalo- equilibration buffer and then the target protein was eluted with genase (HAD) like superfamily. A selection of low but significant 200 mM imidazole in equilibration buffer. The resulting fractions (between 16% and 23%) similar proteins [9–16] was shown in a H. Pan et al. / FEBS Letters 585 (2011) 2545–2550 2547

Fig. 1. Sequence alignment of EH with haloacid dehalogenases and haloacetate dehalogenase. The sequences were aligned using the multiple alignment program ClustalW2 and were shown in order of sequence similarity to EH in this study. Identical amino acids were marked with ‘‘⁄’’, conserved substitution residues with ‘‘:’’ and the residues which were mutagenized in this study with ‘‘#’’. The predicted secondary structure elements of EH, and the determined secondary structures of Hdl, DhlB [9] and L-DEX [10] were shown. a-Helices were shown underlined and b-strands were shown as shaded. Sequences: EH, EH from Rhodococcus opacus ML-0004 [6]; Hdl, L-2-haloacid dehalogenase IVa from Pseudomonas cepacia MBA4 [11]; DehCI, L-2-haloacid dehalogenase I from Pseudomonas sp. strain CBS3 [12]; DehH2, haloacetate dehalogenase H-2 from Moraxella sp. strain B [13]; DhlS5I, L-2-haloacid dehalogenase from Agrobacterium tumefaciens RS5 [14]; L-DEX, L-2-haloacid dehalogenase from Pseudomonas sp. strain YL [15]; DhlB, L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 [16].

ClustalW alignment (Fig. 1). Sequence similarity was also found residue as follows: D by N, Q by E, R by K, and vice versa, S and T with hypothetical protein MMAR_3371 from Mycobacterium mari- by A, Y and W by F, M by L and H by N, except for A by S, resulting num M [17], but it was omitted from the alignment because the in 29 different recombinants. protein had not been studied. We found that the following residues The encoding the mutant enzyme were expressed under were highly conserved: seven aspartate (D18, 25, 60, 80, 115, 152 the control of the tac promoter of pTrc99A in E. coli JM109, result- and 193), two arginine (R55 and 209), two lysine (K70 and 164), ing in production of respective proteins. The mutant enzymes were one histidine (H190), four serine (S28, 81, 156 and 189), two thre- expressed in soluble form (except for W54F, D152 N and R209 K) onine (T22 and 133), three tyrosine (Y163, 170 and 192), four tryp- with different proportions of the totally soluble proteins (up to tophan (W54, 71, 107, 113), one methionine (M186), one 30%). The enzymes were purified by immobilized metal affinity asparagine (N134) and one glutamine residue (Q158). No cysteine chromatography and both wild-type and mutant enzymes exhib- or glutamate residues were conserved. There were two glycine res- ited the same retention time on gel filtration. The purified enzymes idues and many hydrophobic amino acid residues (two alanine, six leucine, one valine, one proline, one phenylalanine, but no isoleu- cine residues) which were completely conserved among these Table 1 sequences. Relative activities of wild-type and mutant EHs.a

Enzyme Relative activity (%) Enzyme Relative activity (%) 3.2. Secondary structure predictions Wild-type 100 ± 3.0 S156A 6.2 ± 0.1 D18 N 0.12 ± 0.004 S189A 97.4 ± 4.3 Since the three-dimensional structures of Hdl, DhlB and L-DEX D25 N 47.5 ± 1.6 T22A 0.13 ± 0.002 were known, we studied their secondary structure elements. The D60 N 57.6 ± 0.9 T133A 4.1 ± 0.08 experimental secondary structure elements of Hdl, DhlB and D80 N 82 ± 1.1 Y163F 76.9 ± 0.3 L-DEX were conserved in the sequence alignment (Fig. 1). This D115 N 18.3 ± 0.6 Y170F 0.14 ± 0.002 D152 N – Y192F 33.2 ± 1.5 suggested that the secondary structure elements of Hdl, DhlB and D193 N 0 W54Fb — L-DEX could be extrapolated to EH. Therefore, secondary structure R55 K 1.1 ± 0.003 W71F 20.8 ± 0.9 predictions were carried out with the programs Porter [18], Jpred R209Kb – W107F 32.1 ± 1.4 [19], PSIPRED [20] and PredictProtein [21]. The results were com- K70R 91 ± 3.7 W113F 3.7 ± 0.1 pared and similar predictions were taken as a consensus (Fig. 1). K164R 0.73 ± 0.03 M186L 7.9 ± 0.4 H190 N 0.055 ± 0.001 N134D 0.062 ± 0.0007 The secondary structure predictions, based on the amino acid se- S28A 22.5 ± 0.3 Q158E 29.2 ± 1.2 quence, gave similar results as that from the alignment data. S81A 67.5 ± 3.0 A188S 1.6 ± 0.07

a The enzyme activity was measured and presented as the mean ± S.E.M. for 3.3. Construction and expression of the EH mutants triplicate experiments. The specific activity of wild-type was 467 ± 14 lmol/min/ mg. According to the results of alignment, we replaced each of the b The enzyme formed inclusion body, and therefore their activities could not be conserved charged and polar amino acid residues with another determined. 2548 H. Pan et al. / FEBS Letters 585 (2011) 2545–2550

Table 2 Kinetic parameters, EE values and predicted secondary structure contents of wild-type and mutant EHs.

-1 Enzyme Km (mM) Kcat (s ) EE value (%) Secondary structure contents (%) Helix Sheet Others Wild-type 47.9 390.4 98.9 ± 4.2 33 17 50 D18 N 52.3 1.13 98 ± 4.2 28 24 48 D193 N – – – 28 24 48 R55 K 154.6 3.8 98.6 ± 2.3 28 25 47 K164R 79.5 2.9 98 ± 2.8 28 24 48 H190 N 82.4 0.23 98.8 ± 3.4 28 24 48 T22A 50.1 0.69 98.5 ± 4.1 27 22 51 Y170F 54.2 0.56 98.9 ± 4.6 27 27 45 N134D 71.8 0.24 99 ± 2.7 27 29 43 A188S 70.3 6.2 99 ± 3.9 33 17 50 were found to be homogeneous upon SDS–PAGE analysis (Supple- These results suggest that an oxygen atom from a water molecule mentary Fig. 1). was first transferred to the enzyme and then to the product.

3.4. Characterization of the mutant enzymes 4. Discussion

The relative activities of the mutant enzymes toward disodium The amino acid sequence of our EH, which belongs to HAD like cis-epoxysuccinate are shown in Table 1. Those mutants, whose superfamily, showed similarity to L-2-haloacid dehalogenases and relative activities were 2% lower than the wild-type enzyme’s rel- haloacetate dehalogenase from different sources (Fig. 1), but it ative activity, were listed as follows: D18 N, D193 N, R55 K, K164R, showed no homology to the EHs from different sources, such as H190 N, T22A, Y170F, N134D and A188S. Kinetic parameter analy- Agrobacterium radiobacter AD1 [22], Corynebacterium sp. C12 [23] sis (Table 2) showed that the Kcat values of these 9 mutants were and Pseudomonas stutzeri A1501 [24], which belong to the esterase significantly lower, but the Km values of the mutants were not so lipase superfamily. According to the result of the secondary struc- high compared with that of the wild-type enzyme. The EE value ture predictions (Fig. 1 and Table 2), we presumed that our EH has of the wild-type enzyme was indistinguishable from those of the an a/b-type structure, but it is different from the a/b hydrolase fold mutant enzymes (Table 2). In addition, both wild-type and mutant which has a common characteristic fold comprising an eight- enzymes exhibited the same retention time on gel filtration. How- stranded parallel b-sheet flanked by a-helices and ever, CD spectra (Supplementary Fig. 2) of these 9 mutant enzymes residues on the topologically and sterically equivalent loci on the (except for A188S) showed slightly differences with the wild-type specific turns and loop: the nucleophile elbow, the acidic turn, enzyme, and the predicted secondary structure contents (accord- and the histidine loop [25]. Thus our EH cannot be grouped into ing to the CD spectra) are shown in Table 2. the same superfamily as the a/b hydrolase fold enzymes. Site-directed mutagenesis experiment showed that the replace- 18 3.5. Single and multiple turnover reactions of wild-type EH in H2 O ment of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss of activity (Table 1), indicating that these Under single turnover conditions, less than 5% tartrate produced residues are probably involved in the catalysis. EE values (Table 18 18 in H2 O contained O(Fig. 2A), whereas under the multiple turn- 2) showed that the replacement of these residues did not change 18 over conditions, more than 95% tartrate contained O(Fig. 2B). the stereospecificity of enzyme. 3D structures of Hdl, L-DEX and DhlB have been reported [9,10,26,27], and Hdl possess the most amino acid sequence identity with EH. Therefore, the 3D structure of our EH was predicted using Hdl as the template. The predicted 3D structure of EH is shown in Fig. 3A. Superposition among these 4 enzymes is shown in Fig. 3B. Hdl, L-DEX and DhlB possess 23%, 19% and 16% amino acid sequence identity with EH, and their structures have a carbon alpha root mean square of 0.4 Å, 1.1 Å and 1.0 Å to EH, indicating that these 4 enzyme have very similar folds. From Fig. 1 and Fig. 3C, we found that some functionally important residues of our EH (D18, D193, R55, K164, H190, T22, Y170, N134 and A188) lie on the topologically equivalent sites to those of the residues of Hdl (D11, D181, R42, K152, N178, T15, Y158, N120 and S176), L-DEX (D10, D180, R41, K151, N177, T14, Y157, N119 and S175) and DhlB (D8, D176, R39, K147, N173, T12, Tyr153, N115 and S171). These findings suggest that these enzymes might be evolutionally related, although this should be carefully confirmed by further studies. The monomer of Hdl, L-DEX and DhlB consist of two domains that are referred to as the core and cap domains (Fig. 3B). The ac- tive site is located in a cleft region between the two domains (Fig. 3A and Fig. 3C). The core domains of Hdl, L-DEX, DhlB and pre- 18 Fig. 2. Ion spray mass spectra of tartrate produced with wild-type EH in H2 O. The dicted EH are structurally similar to a typical HAD superfamily spectra were obtained between 147 and 153 atomic mass units. Step size was 0.1 Rossmannoid fold [27]. However, differences occur in the cap do- atomic mass units and dwell time was 10 ms/step. Ion spray voltage was set at 4 kV and the orifice voltage was optimized at 50 V. A, single turnover reaction; B, mains, which are thought to be responsible for the diversity ob- multiple turnover reaction. served in substrate specificity and reaction mechanisms. H. Pan et al. / FEBS Letters 585 (2011) 2545–2550 2549

Fig. 4. Proposed reaction mechanism for EH from Rhodococcus opacus. In step 1, a covalent ester intermediate is formed between enzyme and substrate by attack of the nucleophile Asp18 on the carbon atoms of the oxirane ring. Presumably, an unidentified proton donor (B-H) assists in ring opening. This enzyme-substrate ester is hydrolyzed in step 2 by a water molecule activated by the His190-Asp193 pair. The role of the amino acid residues presented by the numbers in the catalytic process is suggested by sequence comparison to related proteins (Fig. 1) and verified by the results of the present reports.

structures and reaction mechanisms of Hdl, L-DEX and DhlB have been reported [9,10,26,27], we can accordingly predict functional roles of our catalytic residues. The first step is nucleophilic attack by Asp11 (Asp10, 8, residues will be numbered as they are in Hdl, with L-DEX and DhlB numbering following consecutively in parentheses) on the a-carbon of the substrate molecule, resulting in the displacement of the halide atom and the formation of an es- ter bond between the enzyme and substrate. Therefore, we pre- dicted that Asp18 might act as a nucleophile in our EH. The second step of the reaction involves the hydrolysis of the ester bond by an active site water molecule thereby allowing the release Fig. 3. Predicted 3D structure of EH (A) and superposition in whole structure (B) and in active site structure (C) among predicted EH, Hdl, DhlB and L-DEX. (A) 3D of the reaction product. An active water is tightly held by residues structure of EH is predicted by Swiss-Model software using chain B of Hdl (PDB ID: including Asn178 (Asn177, 173), and Asp181 (Asp180, 176), in a 2NO4) as the template, and viewed with Swiss Pdb-Viewer. The secondary structure suitable position near Asp11 (Asp10, 8) for ester hydrolysis. There- elements are presented as ribbons, colored as a spectrum from N-terminus (blue) to fore, according to topologically equivalent sites (Fig. 3C) and rela- C-terminus (red). 9 catalytically important residues are shown. (B) Structural tive activity assay (Table 1), we assume that our His190 and superposition among predicted EH (red), Hdl (chian B, PDB ID: 2NO4, yellow), DhlB (chian B, PDB ID: 1AQ6, blue) and L-DEX (PDB ID: 1QH9, green). For both (A) and (B), Asp193 may be probably essential for hydrolysis of the ester inter- a-helices are presented by spirals and b-strands by arrows. (C) Superposition in the mediate. Two amino acids of histidine-acid catalytic triad form a active site structure among predicted EH (red), Hdl (yellow), DhlB (blue) and L-DEX classic catalytic triad, which also can be found in other classes of (green). Labeled residues are shown for EH, which lie on the topologically hydrolytic enzymes, such as EH [28]. However, from the crystal equivalent sites to those of the active site residues of Hdl, L-DEX and DhlB. structures of Hdl, L-DEX and DhlB, it can be seen that such a cata- lytic triad is absent, since no histidine is found in their active sites, whereas, such classic catalytic triad (H190-D193) exists in our EH Single and multiple turnover reaction studies (Fig. 2) suggested which is the difference between HAD and our EH. that an oxygen atom from water was incorporated into the product On basis of the analysis above, a two-step catalytic mechanism via incorporation into enzyme. This observation is consistent with characterized by a nucleophile-histidine-acid catalytic triad a two-step mechanism, and the reaction of Hdl, L-DEX and DhlB (Asp18-His190-Asp193) is proposed for our EH (Fig. 4), which is proceeds through this mechanism. Since the three-dimensional similar to the EH from Agrobacterium radiobacter AD1 (Asp107- 2550 H. Pan et al. / FEBS Letters 585 (2011) 2545–2550

Asp246-His275) [29]. These two enzymes proceed through the [7] Pan, H.F., Xie, Z.P., Bao, W.N. and Zhang, J.G. (2008) Optimization of culture same reaction mechanism and preserve the similar arrangement conditions to enhance cis-epoxysuccinate hydrolase production in Escherichia coli by response surface methodology. Biochem. Eng. J. 42, 133–138. of the catalytic residues. Nevertheless, they do not share any signif- [8] Pan, H.F., Bao, W.N., Xie, Z.P., Zhang, J.G. and Li, Y.Q. (2010) Immobilization of icant sequence similarity nor do they have similar secondary struc- Escherichia coli cells with cis-epoxysuccinate hydrolase activity for D(–)- ture, which suggests a divergent evolution of this protein. tartaric acid production. Biotechnol. Lett. 32, 235–241. [9] Ridder, I.S., Rozeboom, H.J., Kalk, K.H., Janssen, D.B. and Dijkstra, B.W. (1997) Thr15 (Thr14, 12) is the main residue of since its Three-dimensional structure of L-2-haloacid dehalogenase from Xanthobacter c1 d1 O atom is tightly hydrogen-bonded with O of Asp11 (Asp10, 8), autotrophicus GJ10 complexed with the substrate-analogue formate. J. Biol. while Lys152 (Lys151, 147) is weakly hydrogen-bonded with the Chem. 272, 33015–33022. d2 [10] Hisano, T., Hata, Y., Fujii, T., Liu, J.Q., Kurihara, T., Esaki, N. and Soda, K. (1996) esterified oxygen O of the Asp11 (Asp10, 8), indicating that our Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. An Thr22 and Lys164 probably play similarly indispensable roles. alpha/beta hydrolase structure that is different from the alpha/beta hydrolase The Od1 of Asp11 (Asp10, 8) is also stabilized by hydrogen bonding fold. J. Biol. Chem. 271, 20322–20330. c [11] Murdiyatmo, U., Asmara, W., Tsang, J.S., Baines, A.J., Bull, A.T. and Hardman, with O of Ser176 (Ser175, 171). However, the corresponding site D.J. (1992) Molecular biology of the 2-haloacid halidohydrolase IVa from to our enzyme is Ala188, which suggested the differences between Pseudomonas cepacia MBA4. Biochem. J. 284, 87–93. HAD and our EH. Therefore we replaced Ala by Ser on 188 site and [12] Schneider, B., Muller, R., Frank, R. and Lingens, F. (1991) Complete nucleotide the mutant A188S’s relative activity is 2% lower than the wild-type sequences and comparison of the structural genes of two 2-haloalkanoic acid dehalogenases from Pseudomonas sp. strain CBS3. J. Bacteriol. 173, 1530–1535. enzyme’s relative activity (Table 1). [13] Kawasaki, H., Tsuda, K., Matsushita, I. and Tonomura, K. (1992) Lack of Arg42 (Arg41, 39), positioned in the entrance to the active site, homology between two haloacetate dehalogenase genes encoded on a may regulate substrate entry and exit, possibly through interac- plasmid from Moraxella sp. Strain B. J. Gen. Microbiol. 138, 1317–1323. [14] Kohler, R., Brokamp, A., Schwarze, R., Reiting, R.H. and Schmidt, F.R. (1998) tions with substrate/product carboxyl groups. Although the spe- Characteristics and DNA-sequence of a cryptic haloalkanoic acid dehalogenase cific activity of mutant N119D of L-DEX (92%) is almost equal to from Agrobacterium tumefaciens RS5. Curr. Microbiol. 36, 96–101. the wild-type enzyme, crystallographic study shows that Asn120 [15] Nardi-Dei, V., Kurihara, T., Okamura, T., Liu, J.Q., Koshikawa, H., Ozaki, H., Terashima, Y., Esaki, N. and Soda, K. (1994) Comparative studies of genes (Asn119, 115) locates around the active site and its main-chain encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. amido nitrogen is hydrogen-bonded with the carboxyl oxygen of Strain YL, other dehalogenases, and two related hypothetical proteins from substrate. Therefore, we assumed that our Arg55 and Asn134 Escherichia coli. Appl. Environ. Microbiol. 60, 3375–3380. [16] van der Ploeg, J., van Hall, G. and Janssen, D.B. (1991) Characterization of the may serve as the recognition sites for the substrate carboxyl haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing groups. of the dhlB gene. J. Bacteriol. 173, 7925–7933. In conclusion, residues D18, D193, R55, K164, H190, T22, Y170, [17] Stinear, T.P., Seemann, T., Harrison, P.F., Jenkin, G.A., Davies, J.K., Johnson, P.D., Abdellah, Z., Arrowsmith, C., Chillingworth, T., Churcher, C., Clarke, K., Cronin, N134 and A188 play indispensable roles in the catalytic function of A., Davis, P., Goodhead, I., Holroyd, N., Jagels, K., Lord, A., Moule, S., Mungall, K., our EH, such as in nucleophilic attack, substrate binding and acti- Norbertczak, H., Quail, M.A., Rabbinowitsch, E., Walker, D., White, B., vation of water, and the enzyme reaction proceeded through the Whitehead, S., Small, P.L., Brosch, R., Ramakrishnan, L., Fischbach, M.A., two-step mechanism involving the formation of a covalent inter- Parkhill, J. and Cole, S.T. (2008) Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. mediate. Further crystallographic study of the enzyme is now Genome Res. 18, 729–741. being carried out to reveal the roles of these catalytic residues in [18] Pollastri, G. and McLysaght, A. (2005) Porter: a new, accurate server for more detail. protein secondary structure prediction. Bioinformatics 21, 1719–1720. [19] Cole, C., Barber, J.D. and Barton, G.J. (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201. Acknowledgements [20] Jones, D.T. (1999) Protein secondary structure prediction based on position- specific scoring matrices. J. Mol. Biol. 292, 195–202. [21] Rost, B., Yachdav, G. and Liu, J. (2004) The PredictProtein Server. Nucleic Acids This work was funded by Hangzhou Bioking Biochemical Engi- Res. 32, W321–W326. neering Co., Ltd. [22] Rink, R., Fennema, M., Smids, M., Dehmel, U. and Janssen, D.B. (1997) Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacter AD1. J. Biol. Chem. 272, 14650–14657. 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