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Indexed by:期刊论文

Date of Publication:2014-06-10

Journal:ENZYME AND MICROBIAL TECHNOLOGY

Included Journals:EI、PubMed、SCIE、Scopus

Volume:60

Issue:60

Page Number:72-79

ISSN No.:0141-0229

Key Words:Characterization; Glyoxylate reductase; Paecilomyes thermophila; Three-dimensional structure; Coenzyme specificity

Abstract:A glyoxylate reductase gene (PtGR) from the fungus Paecilomyces thennophila was cloned and expressed in Escherichia coli. PtGR was biochemically and structurally characterized. PtGR has an open reading frame of 993 bp encoding 330 amino acids. The deduced amino acid sequence has low similarities to the reported glyoxylate reductases. The purified PtGR forms a homodimer. PtGR displayed an optimum pH of 7.5 and broad pH stability (pH 4.5-10). It exhibited an optimal temperature of 50 degrees C and was stable up to 50 degrees C. PtGR was found to be highly specific for glyoxylate, but it showed no detectable activity with 4-methyl-2-oxopentanoate, phenylglyoxylate, pyruvate, oxaloacetate and alpha-ketoglutarate. PtGR prefered NADPH rather than NADH as an electron donor. Moreover, the crystal structure of PtGR was determined at 1.75 A resolution. The overall structure of apo-PtGR monomer adopts the typical n-2-hydroxy-acid dehydrogenase fold with a "closed" conformation unexpectedly. The coenzyme specificity is provided by a cationic cluster consisting of N184, R185, and N186 structurally. These structural observations could explain its different coenzyme and substrate specificity. (C) 2014 Elsevier Inc. All rights reserved.