Site-directed mutagenesis of the amino acid residues in β-strand III [Val 30-Val 36] of d-amino acid aminotransferase of Bacillus sp. YM-1

• Published in: FEBS letters 1996-12, Vol.398 (2), p.141-145
• Main Authors: Ro, Hyeon-Su, Hong, Seung-Pyo, Seo, Hwa-Jung, Yoshimura, Tohru, Esaki, Nobuyoshi, Soda, Kenji, Kim, Hak-Sung, Sung, Moon-Hee
• Format: Article
• Language: English
• Subjects: Alanine Transaminase - chemistry; Alanine Transaminase - genetics; Alanine Transaminase - metabolism; Bacillus; Bacillus - enzymology; Bacillus - genetics; Binding Sites; D-Alanine Transaminase; d-Amino acid aminotransferase; Glutamic Acid - chemistry; Hydrogen Bonding; Hydrogen-Ion Concentration; Keto Acids - metabolism; Ketoglutaric Acids - metabolism; Kinetics; Lysine - chemistry; Mutagenesis, Site-Directed; pH titration; Pyridoxal 5′-phosphate; Pyridoxal Phosphate - metabolism; Site-directed mutagenesis; Spectrometry, Fluorescence; Substituted aldamine; Valine - chemistry
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(96)01222-7

The β-strand III formed by amino acid residues Val 30-Val 36 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 (Tyr 31, Val 33, Lys 35) in the strand are revealed to be directed toward the active site. Interestingly, Glu 32 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 Lys 35 interacted electrostatically with the negative charge at the side chain of α-ketoglutarate. Site-directed mutagenesis of the Glu 32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu 32-substituted enzymes, the Glu 32 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 Lys 145 residue.