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Title 

Site-directed mutagenesis of the amino acid residues in β-strand III [Val30-Val36] of D-amino acid aminotransferase of Bacillus sp. YM-1

Authors 

Hyeon Su RoSeung Pyo HongHwa-Jung SeoTohru YoshimuraNobuyoshi EsakiKenji SodaHak-Sung KimMoon Hee Sung

Publisher 

Elsevier

Issue Date 

1996

Citation 

FEBS Letters, vol. 398, no. 0, pp. 141-145

Keywords 

D-Amino acid aminotransferasepH titrationPyridoxal 5'-phosphateSite-directed mutagenesisSubstituted aldamineaminotransferasebacillusenzyme active siteenzyme activitysite directed mutagenesis

Abstract 

The β-strand III formed by amino acid residues Val30-Val36 is located across the active site of the thermostable D-amino acid aminotransferase (D-AAT) from thermophilic Bacillus sp. YM-1, and the odd-numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in k(cat) value, retaining less than 1% of the wild-type enzyme activity. The k(cat) values of V33A and K35A were changed slightly, but the K(m) of K35A for α-ketoglutarate was increased to 35.6 mM, compared to the K(m) value of 2.5 mM for the wild-type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of α-ketoglutarate. Site-directed mutagenesis of the Glu32 residue was conducted to demtrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32- substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5'-phosphate (PLP) and the ε-amino group of the Lys145 residue.

ISSN 

0014-5793

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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