Substitutability of metal-binding sites in an alcohol dehydrogenase.

Yuexin BI; Yingying Jiang; Zongmin Qin; Ge QU; Zhoutong Sun

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Substitutability of metal-binding sites in an alcohol dehydrogenase.

Author: Yuexin BI ¹ ; Yingying JIANG ² ; Zongmin QIN ² ; Ge QU ² ; Zhoutong SUN ²
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1. School of Life Sciences, University of Science and Technology of China, Hefei 230026, Anhui, China.
2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Publication Type:Journal Article
Keywords: biocatalysis; enzyme engineering; metal-binding sites; metalloenzyme; rational design
MeSH: Alcohol Dehydrogenase/metabolism*; Catalytic Domain; Ligands; Protein Domains; Zinc/metabolism*
From: Chinese Journal of Biotechnology 2022;38(4):1518-1526
CountryChina
Language:Chinese
Abstract: Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.