The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

• Published in: FEMS microbiology letters Vol. 243; no. 1; pp. 157 - 163
• Main Authors: Fundoiano-Hershcovitz, Yifat, Rabinovitch, Larisa, Shulami, Smadar, Reiland, Vera, Shoham, Gil, Shoham, Yuval
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01.02.2005; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Amino Acid Sequence; Aminopeptidase; Aminopeptidases - chemistry; Aminopeptidases - genetics; Aminopeptidases - isolation & purification; Aminopeptidases - metabolism; Aspartic endopeptidase; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacillus subtilis aminopeptidase; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Catalytic mechanism; Cloning, Molecular; E coli; Fundamental and applied biological sciences. Psychology; Genetics; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Protease; Protease inhibitors; Proteinase inhibitors; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Serine; Serine proteinase; Substrate Specificity; Substrates; ywad gene; Zinc - metabolism; Zinc chloride; Zn-metalloenzyme; Zn-metalloenzyme; ywad gene; Bacillus subtilis aminopeptidase; Substrate specificity; Catalytic mechanism; gene; aminopeptidase; Purification; Enzyme; Bacillus subtilis; Aminopeptidases; Sequence alignment; Bacillaceae; Zinc; Peptidases; Glutamyl aminopeptidase; Bacillales; Gene; Bacteria; Hydrolases; Recombinant protein
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/j.femsle.2004.12.001

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 °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. ZnCl 2 was able to restore activity, and the binding stochiometry 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 k cat/ K m values of 1–5 × 10 1 s −1 mM −1.