Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B. subtilis as an active complex with its 23-kDa propeptide.
The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable. We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600. It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease. The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain. We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species. The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity. WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1. Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A. The pH dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant.
Babé
L M
LM
Axys Pharmaceuticals Inc., 180 Kimball Way, South San Francisco, CA 94080, USA. lilia_babe@axyspharm.com
Schmidt
B
B
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011480:Protease Inhibitors; D011498:Protein Precursors; D012697:Serine Endopeptidases; C104015:WprA protein, Bacillus subtilis