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

doi:10.21203/rs.3.rs-3437865/v1

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

https://doi.org/10.21203/rs.3.rs-3437865/v1

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published 12 May, 2024

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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 Zn²⁺, Mn²⁺ 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. ¹⁸O-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

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.

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 H₂¹⁸O 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 H₂¹⁸O, 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 H₂¹⁸O 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).

Characterization of TESH

The optimum temperature and pH of TESH from P. koreensis BK-9 were 45°C and pH 9.0, respectively (Fig. 1A, 1B). 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. 1A, 1C), indicating poor temperature and pH stability.

Insert Fig. 1

Statistical analysis indicated that Mg²⁺, Ca²⁺ were the activators of the enzyme, increased TESH activity significantly (P ＜ 0.001). Zn²⁺and Mn²⁺ 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. 1D). 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. 3A). 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. 3B).

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
^aThe 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 H₂¹⁸O

The single and multiple turnover reactions of TESH from P. koreensis BK-9 in H₂¹⁸O showed that most of the tartrate was ¹⁶O-labeled in the single turnover reaction (Fig. 4A), while most of the tartrate was ¹⁸O-labeled in the multiple turnover reaction (Fig. 4B). 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. 5A), 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. 5B.

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. 5C). 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. 5D). H34 have polar contacts with D104 and Y211 (Fig. 5E). 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 Zn²⁺-dependent, one-step mechanism (Dong et al. 2018).

Insert Fig. 6

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 Zn²⁺, Mn²⁺ 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.

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.

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You are reading this latest preprint version

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

Status:

Journal Publication

Version 1

Abstract

Objectives

Results

Conclusions

Figures

Introduction

Materials and methods

Strains, plasmids, primers, and culture conditions

Multiple sequence alignment and homology modeling

Construction of site-directed mutagenesis

Enzyme purification and characterization

Isotope label

Enzyme assay

Results and discussion

Characterization of TESH

Sequence comparison of TESH and reported EHs

Homology modeling of TESH

Site-directed mutagenesis

Single and multiple turnover reaction of wild-type TESH in H₂¹⁸O

Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

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

Status:

Journal Publication

Version 1

Abstract

Objectives

Results

Conclusions

Figures

Introduction

Materials and methods

Strains, plasmids, primers, and culture conditions

Multiple sequence alignment and homology modeling

Construction of site-directed mutagenesis

Enzyme purification and characterization

Isotope label

Enzyme assay

Results and discussion

Characterization of TESH

Sequence comparison of TESH and reported EHs

Homology modeling of TESH

Site-directed mutagenesis

Single and multiple turnover reaction of wild-type TESH in H218O

Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Single and multiple turnover reaction of wild-type TESH in H₂¹⁸O