Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)

Abstract A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydrox...

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Autores principales:	Zhiguo Wang, Shuo Zhou, Shuangling Zhang, Sa Zhang, Fangmeng Zhu, Xiaolu Jin, Zhenming Chen, Xiaoling Xu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9231e0b840834125b00578885c87fc8a
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spelling	oai:doaj.org-article:9231e0b840834125b00578885c87fc8a2021-12-02T12:32:03ZSemi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)10.1038/s41598-017-03947-82045-2322https://doaj.org/article/9231e0b840834125b00578885c87fc8a2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03947-8https://doaj.org/toc/2045-2322Abstract A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired. In this work, we determined the crystal structure of Tm1743 in complex with its cofactor NADP+ at 2.0 Å resolution, and investigated the enantioselectivity of Tm1743 through semi-rational enzyme design. Molecular simulations based on the crystal structure obtained two binding models representing the pro-S and pro-R conformations of EOPB. Saturation mutagenesis studies revealed that Trp21 and Trp86 play important roles in determining the enantioselectivity of Tm1743. The best (R)- and (S)-EHPB preferring Tm1743 mutants, denoted as W21S/W86E and W21L/W118H, were identified; their ee values are 99.4% and 99.6% and the catalytic efficiencies are 0.81 and 0.12 mM−1s−1, respectively. Our work presents an efficient strategy to improve the enantioselectivity of a natural biocatalyst, which will serve as a guide for further exploration of new green catalysts for asymmetric reactions.Zhiguo WangShuo ZhouShuangling ZhangSa ZhangFangmeng ZhuXiaolu JinZhenming ChenXiaoling XuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Zhiguo Wang Shuo Zhou Shuangling Zhang Sa Zhang Fangmeng Zhu Xiaolu Jin Zhenming Chen Xiaoling Xu Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
description	Abstract A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired. In this work, we determined the crystal structure of Tm1743 in complex with its cofactor NADP+ at 2.0 Å resolution, and investigated the enantioselectivity of Tm1743 through semi-rational enzyme design. Molecular simulations based on the crystal structure obtained two binding models representing the pro-S and pro-R conformations of EOPB. Saturation mutagenesis studies revealed that Trp21 and Trp86 play important roles in determining the enantioselectivity of Tm1743. The best (R)- and (S)-EHPB preferring Tm1743 mutants, denoted as W21S/W86E and W21L/W118H, were identified; their ee values are 99.4% and 99.6% and the catalytic efficiencies are 0.81 and 0.12 mM−1s−1, respectively. Our work presents an efficient strategy to improve the enantioselectivity of a natural biocatalyst, which will serve as a guide for further exploration of new green catalysts for asymmetric reactions.
format	article
author	Zhiguo Wang Shuo Zhou Shuangling Zhang Sa Zhang Fangmeng Zhu Xiaolu Jin Zhenming Chen Xiaoling Xu
author_facet	Zhiguo Wang Shuo Zhou Shuangling Zhang Sa Zhang Fangmeng Zhu Xiaolu Jin Zhenming Chen Xiaoling Xu
author_sort	Zhiguo Wang
title	Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
title_short	Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
title_full	Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
title_fullStr	Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
title_full_unstemmed	Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)
title_sort	semi-rational engineering of a thermostable aldo–keto reductase from thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (ehpb)
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/9231e0b840834125b00578885c87fc8a
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Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)

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