反式环氧琥珀酸水解酶生产中酒石酸的必需氨基酸残基和催化机理。

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2024-10-01 Epub Date: 2024-05-13 DOI:10.1007/s10529-024-03490-3

Hongxiu Liao, Haifeng Pan, Jinfeng Yao, Ronglin Zhu, Wenna Bao

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引用次数: 0

摘要

研究目的本研究旨在探讨韩国假单胞菌反式环氧琥珀酸水解酶生产中酒石酸的必需氨基酸残基和催化机理：该酶的最佳催化条件分别为 45 ℃ 和 pH 9.0。它受到 Zn2+、Mn2+ 和 SDS 的强烈抑制。Michaelis-Menten 酶动力学分析表明，Km 值为 3.50 mM，kcat 为 99.75 s-1，EE 值超过 99.9%。多重序列比对和同源建模显示，该酶属于 MhpC 超家族，具有典型的 α/β 水解酶折叠结构。定点突变表明，H34、D104、R105、R108、D128、Y147、H149、W150、Y211 和 H272 是重要的催化残基。18O标记研究表明，该酶通过两步催化机制发挥作用：首次报道了反式环氧琥珀酸水解酶的结构和催化机理。结论：首次报道了反式环氧琥珀酸水解酶的结构和催化机理，其中有 10 个残基对其催化起关键作用，并提出了由 Asp-His-Asp 三元催化组成的两步催化机理。

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

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

Objectives: This study aimed to discuss the essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase from Pseudomonas koreensis for the 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, with an exceptional EE value exceeding 99.9%. Multiple sequence alignment and homology modeling revealed that the enzyme belonged to MhpC superfamily and possessed 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. The ¹⁸O-labeling study suggested the enzyme acted via two-step catalytic mechanism.

Conclusions: The structure and catalytic mechanism of trans-epoxysuccinate hydrolase were first reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad was proposed.

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.