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Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159

D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furth...

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Autores principales: Gao, Xiuzhen, Ma, Qinyuan, Song, Huihui, Sun, Xinming, Li, Zhiyun, Liu, Mingfei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084900/
https://www.ncbi.nlm.nih.gov/pubmed/32150965
http://dx.doi.org/10.3390/ijms21051788
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author Gao, Xiuzhen
Ma, Qinyuan
Song, Huihui
Sun, Xinming
Li, Zhiyun
Liu, Mingfei
author_facet Gao, Xiuzhen
Ma, Qinyuan
Song, Huihui
Sun, Xinming
Li, Zhiyun
Liu, Mingfei
author_sort Gao, Xiuzhen
collection PubMed
description D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furthermore, StDAPDH shows excellent thermostability compared to other meso-DAPDHs. However, the cofactor of StDAPDH is NADP(H), which is less common than NAD(H) in industrial applications. Therefore, cofactor engineering for StDAPDH is needed. In this study, the highly conserved cofactor binding sites around the adenosine moiety of NADPH were targeted to determine cofactor specificity. Lysine residues within a loop were found to be critical for the cofactor specificity of StDAPDH. Replacement of lysine with arginine resulted in the activity of pyruvic acid with NADH as the cofactor. The affinity of K159R to pyruvic acid was equal with NADH or NADPH as the cofactor, regardless of the mutation. Molecular dynamics simulations revealed that the large steric hindrance of arginine and the interaction of the salt bridge between NADH and arginine may have restricted the free movement of NADH, which prompted the formation of a stable active conformation of mutant K159R. These results provide further understanding of the catalytic mechanism of StDAPDH and guidance for the cofactor engineering of StDAPDH.
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spelling pubmed-70849002020-03-23 Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159 Gao, Xiuzhen Ma, Qinyuan Song, Huihui Sun, Xinming Li, Zhiyun Liu, Mingfei Int J Mol Sci Article D-amino acid production from 2-keto acid by reductive amination is an attractive pathway because of its high yield and environmental safety. StDAPDH, a meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum, was the first meso-DAPDH to show amination of 2-keto acids. Furthermore, StDAPDH shows excellent thermostability compared to other meso-DAPDHs. However, the cofactor of StDAPDH is NADP(H), which is less common than NAD(H) in industrial applications. Therefore, cofactor engineering for StDAPDH is needed. In this study, the highly conserved cofactor binding sites around the adenosine moiety of NADPH were targeted to determine cofactor specificity. Lysine residues within a loop were found to be critical for the cofactor specificity of StDAPDH. Replacement of lysine with arginine resulted in the activity of pyruvic acid with NADH as the cofactor. The affinity of K159R to pyruvic acid was equal with NADH or NADPH as the cofactor, regardless of the mutation. Molecular dynamics simulations revealed that the large steric hindrance of arginine and the interaction of the salt bridge between NADH and arginine may have restricted the free movement of NADH, which prompted the formation of a stable active conformation of mutant K159R. These results provide further understanding of the catalytic mechanism of StDAPDH and guidance for the cofactor engineering of StDAPDH. MDPI 2020-03-05 /pmc/articles/PMC7084900/ /pubmed/32150965 http://dx.doi.org/10.3390/ijms21051788 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gao, Xiuzhen
Ma, Qinyuan
Song, Huihui
Sun, Xinming
Li, Zhiyun
Liu, Mingfei
Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title_full Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title_fullStr Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title_full_unstemmed Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title_short Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159
title_sort altered cofactor preference of thermostable stdapdh by a single mutation at k159
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084900/
https://www.ncbi.nlm.nih.gov/pubmed/32150965
http://dx.doi.org/10.3390/ijms21051788
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