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A Study on Substrate Selectivity of AKR5C3 Based on the Analysis of Crystal Structure

Author: LiuXu
Tutor: WeiDongZhi
School: East China University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Gluconobacter oxydans aldo-keto reductase (AKR) crystal structure site-directed mutagenesis
CLC: Q55
Type: Master's thesis
Year: 2011
Downloads: 101
Quote: 0
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Abstract


Gox0644 was shown a broad substrate spectrum in the reduction of different aldehydes a-diketones,a-keto esters and 2,5-diketogluconate. Like2,5-DKG reductase, Gox0644 could catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid, a key precursor to L-ascorbic acid (vitamin C). These examples indicated that Gox0644 is an attractive biocatalyst with a considerable application potential. A cytosolic NADPH-dependent carbonyl reductase from Gluconobacter oxydans, Gox0644, was designated AKR5C3 according to the nomenclature system provided by the group of Trevor Penning.In this study, we present the crystal structures of a 2,5-diketogluconate reductase Gox0644 (AKR5C3) complexed with NADP(H) and substrate. Structural comparison of the Gox0644 structures with other known AKR structures supplemented by catalytic assay confirmed the conserved catalytic tetrad of Asp53, Tyr58, Lys79 and His112 responsible for substrate catalysis, whereas unique Trp191, Arg 192 and Lys234 responsible for NADP(H) binding. Considering the characteristic of amino acids and the senior structure of protein crystal, rational design was performed.The soluble mutant proteins were obtained from the E.coli BL21. A Gox0644 mutant D53A obtained by structure-based screening exhibited high substrate selection ability and elevated catalytic activity. Crystal structure of D53A mutant revealed the position shift of the bound NADP(H), therefore the reshaping of the substrate-binding pocket, plays the determination role to improve the substrate selection ability. Another R192G mutant showed significant increasing activity especially with hydrophobic substrates. The discovery of the multiple functional roles of the conserved Asp and Arg residues of AKR family proteins suggests a new strategy to engineer aldo-keto reductases with high activity for industrial application.

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