The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase.
The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).
Fundoiano-Hershcovitz
Yifat
Y
Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Rabinovitch
Larisa
L
Shulami
Smadar
S
Reiland
Vera
V
Shoham
Gil
G
Shoham
Yuval
Y
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000626:Aminopeptidases; D015032:Zinc