Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning.
The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.
Saito
Makoto
M
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Tokyo, Japan.
Nishimura
Katsushi
K
Hasegawa
Yuichiro
Y
Shinohara
Tomomitsu
T
Wakabayashi
Sadao
S
Kurihara
Tatsuo
T
Ishizuka
Morio
M
Nagata
Yoko
Y
D000409:Alanine / Q000378:metabolism; D000411:Alanine Racemase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight
D000411:Alanine Racemase; D000409:Alanine