Site-directed mutagenesis of the amino acid residues in beta-strand III [Val30-Val36] of D-amino acid aminotransferase of Bacillus sp. YM-1.
The beta-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 Km of K35A for alpha-ketoglutarate was increased to 35.6 mM, compared to the Km 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 alpha-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 epsilon-amino group of the Lys145 residue.
Ro
H S
HS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Hong
S P
SP
Seo
H J
HJ
Yoshimura
T
T
Esaki
N
N
Soda
K
K
Kim
H S
HS
Sung
M H
MH
D000410:Alanine Transaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D051263:D-Alanine Transaminase; D018698:Glutamic Acid / Q000737:chemistry; D045782:Hemiterpenes; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014633:Valine / Q000737:chemistry
D045782:Hemiterpenes; D007651:Keto Acids; D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; C001505:alpha-ketoisovalerate; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase; D014633:Valine; D008239:Lysine