Characterization of endogenous pyridoxal 5'-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4.
An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX(OF4)), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadX(OF4) was purified to homogeneity by His(6)-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 degrees C and the optimal pH was 10.5. The kinetic parameters K(m) and V(max) at 40 degrees C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.
Ju
Jiansong
J
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Xu
Shujing
S
Wen
Jianxin
J
Li
Gang
G
Ohnishi
Kouhei
K
Xue
Yanfen
Y
Ma
Yanhe
Y
D000409:Alanine / Q000378:metabolism; D000411:Alanine Racemase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011732:Pyridoxal Phosphate; D000411:Alanine Racemase; D000409:Alanine