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Updated: May 27, 2026
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Expanding the cofactor range of aldehyde reductase YqhD by mutating key amino acids
Xiaohui Wu1,2, Yinghan Hu1,2, Yanzhe Huang1,2
1Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, PR China.
Abstract:
YqhD is a broad-substrate aldehyde reductase that natively prefers NADPH and is widely used in alcohol biosynthesis. However, insufficient NADPH supply and low cofactor regeneration efficiency limit its catalytic performance. To address this limitation, we engineered YqhD through structure-guided semi-rational design and obtained a mutant G37D with markedly improved dual-cofactor compatibility with NADPH and NADH. With butanal and 3-hydroxypropionaldehyde as substrates, G37D exhibited 2.6- and 4.8-fold increases in catalytic efficiency (kcat/Km) with NADH as the cofactor, while retaining > 70% of the wild-type efficiency with NADPH, resulting in comparable efficiencies with both cofactors. Further structural analysis and molecular dynamics simulations revealed that the G37D mutation enhances dual-cofactor utilization by reprogramming the hydrogen-bond network and stabilizing the catalytically favorable closed conformation. This study provides an improved enzyme module for the biomanufacturing of 1,3-propanediol and related alcohols, and offers new insights into the design of versatile catalysts for diverse biocatalytic scenarios.
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