A recombinant L-threonine aldolase with high catalytic efficiency for the asymmetric synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2024-12-10 DOI:10.1007/s10529-024-03553-5

Bijing Lei, Wan Jiang, Jinsong Ma, Caiyun Wang, Yinping Pan, Zhi Zhang, Bochu Wang, Jian Guo, Na Qi

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引用次数: 0

Abstract

Objectives: To develop robust variants of L-threonine aldolases (L-TAs), potent catalysts for synthesizing asymmetric β-hydroxy-α-amino acids, it is necessary to identify critical residues beyond the known active site residues.

Results: Through virtual screening, a neglected residue Asn305, was identified as critical for catalytic efficiency. Subsequent site-saturation mutagenesis led to a potent variant N305R which exhibited excellent conversions of 88%_conv (87%_de) and 80%_conv (94%_de) for the synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine respectively. This variant not only outperformed the template enzyme, but also represented a promising L-TA for synthesizing the two β-hydroxy-α-amino acids. It was suggested that Arg305 of the variant N305R generated strong cation-arene interaction and electrostatic force with the intermediates, leading to strengthened binding, enhanced L-threo favored orientation and wider entrance.

Conclusions: Our work not only provided an excellent variant N305R, but also suggested the crucial function of a neglected residue Asn305, which offered valuable experiences for other L-TA studies.

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一种高效催化不对称合成l -三苯基丝氨酸和l -四氟苯基丝氨酸的重组l -苏氨酸醛缩酶。

目的：l -苏氨酸醛缩酶（L-TAs）是合成不对称β-羟基-α-氨基酸的有效催化剂，为了开发其强大的变体，有必要鉴定已知活性位点残基之外的关键残基。结果：通过虚拟筛选，一个被忽视的残留物Asn305被确定为催化效率的关键。随后的位点饱和诱变导致了一个强效变异N305R，它在合成l -三苯基丝氨酸和l -三苯基-4氟丝氨酸方面分别表现出88%的转化率（87%de）和80%的转化率（94%de）。该变体不仅优于模板酶，而且代表了一种有前途的L-TA用于合成两种β-羟基-α-氨基酸。结果表明，突变体N305R的Arg305与中间体产生强烈的阳离子-芳烃相互作用和静电力，导致结合增强，l -三o取向增强，入口更宽。结论：我们的工作不仅提供了一个优秀的变异N305R，而且揭示了一个被忽视的残基Asn305的关键功能，为其他L-TA研究提供了宝贵的经验。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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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.