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

FEBS Letters

Ro, Hyeon-Su²  Seo, Hwa-Jung³  Sung, Moon-Hee³  Hong, Seung-Pyo³  Yoshimura, Tohru¹  Esaki, Nobuyoshi¹  Kim, Hak-Sung²  Soda, Kenji¹ 

[1] Institute for Chemical research, Kyoto University, Uji, Kyoto 611, Japan;Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305–701, South Korea;Microbial Conversion Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusong-gu, Taejon 305–600, South Korea

关键词: d-Amino acid aminotransferase;    Site-directed mutagenesis;    Substituted aldamine;    pH titration;    Pyridoxal 5′-phosphate;   

DOI  :  10.1016/S0014-5793(96)01222-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 demonstrate 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.

【 授权许可】

Unknown