基于功能环工程改善羰基还原酶的催化性能。

IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Tao-Shun Zhou, Xiang-Yang Li, Xiao-Jian Zhang, Xue Cai, Zhi-Qiang Liu, Yu-Guo Zheng
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引用次数: 0

摘要

维贝琼是一种强效、选择性的β3-肾上腺素能受体激动剂,其手性前体(2S,3R)-氨基羟基酯(1b)对其合成至关重要。本研究将环路工程学应用于藻类Exiguobacterium的羰基还原酶(EaSDR6),以实现(rac)-氨基酮酯1a的不对称还原。与野生型 EaSDR6(WT)相比,变体 M5(A138L/A190V/S193A/Y201F/N204A)的催化效率提高了 868 倍(kcat/Km = 260.3 s-1 mM-1),并对目标产物 1b 具有理想的立体选择性(对映体过量率 >99%,e.e.;非对映体过量率 >99%,d.e.)。与 WT 的结构比对表明,137-154 环和 182-210 环可能在促进催化和底物结合方面起着重要作用。此外,WT-1a 和 M5-1a 复合物的分子动力学(MD)模拟表明,M5-1a 表现出更有效的亲核攻击距离,更容易进入预反应状态。结合自由能的计算分析表明,与 WT 相比,M5 对底物 1a 的亲和力更强,这与其 Km 值的降低是一致的。在有机-水双相条件下,M5 突变体在底物 1a 为 300 g/L 的情况下,12 小时内的转化率大于 99%(据报道,底物负载量最高)。这项研究通过功能环工程提高了羰基还原酶的催化性能,并为大规模生物合成维贝琼中间体建立了一个稳健的框架。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Improving the catalytic performance of carbonyl reductase based on the functional loops engineering.

Improving the catalytic performance of carbonyl reductase based on the functional loops engineering.

Vibegron functions as a potent and selective β3-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (kcat/Km = 260.3 s-1 mM-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.

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来源期刊
Biotechnology and Bioengineering
Biotechnology and Bioengineering 工程技术-生物工程与应用微生物
CiteScore
7.90
自引率
5.30%
发文量
280
审稿时长
2.1 months
期刊介绍: Biotechnology & Bioengineering publishes Perspectives, Articles, Reviews, Mini-Reviews, and Communications to the Editor that embrace all aspects of biotechnology. These include: -Enzyme systems and their applications, including enzyme reactors, purification, and applied aspects of protein engineering -Animal-cell biotechnology, including media development -Applied aspects of cellular physiology, metabolism, and energetics -Biocatalysis and applied enzymology, including enzyme reactors, protein engineering, and nanobiotechnology -Biothermodynamics -Biofuels, including biomass and renewable resource engineering -Biomaterials, including delivery systems and materials for tissue engineering -Bioprocess engineering, including kinetics and modeling of biological systems, transport phenomena in bioreactors, bioreactor design, monitoring, and control -Biosensors and instrumentation -Computational and systems biology, including bioinformatics and genomic/proteomic studies -Environmental biotechnology, including biofilms, algal systems, and bioremediation -Metabolic and cellular engineering -Plant-cell biotechnology -Spectroscopic and other analytical techniques for biotechnological applications -Synthetic biology -Tissue engineering, stem-cell bioengineering, regenerative medicine, gene therapy and delivery systems The editors will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science. Submission of papers dealing with routine aspects of bioprocessing, description of established equipment, and routine applications of established methodologies (e.g., control strategies, modeling, experimental methods) is discouraged. Theoretical papers will be judged based on the novelty of the approach and their potential impact, or on their novel capability to predict and elucidate experimental observations.
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