title: Extracellular autoprocessing of a metalloprotease from Streptomyces cacaoi.
abstract: We have previously demonstrated that the extracellular neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a 60-kDa preproenzyme (P60), then processed to the 35-kDa mature form (P35) (Chang, P. C., Kuo, T.-C., Tsugita, A., and Lee, Y.-H. W. (1990) Gene (Amst.) 88, 87-95). In this study, we investigated the active site and the mechanism involved in the maturation of the protease. Site-specific mutations at the putative zinc-binding ligands and active site of Npr at His202, Glu203, His206, and Glu240 led to complete abolishment of Npr activity and concomitant accumulation of a 57-kDa inactive protein (P57) which was secreted. Sequence analysis of the NH2 terminus indicated that P57 was derived from P60 after removal of the signal peptide and represented the proenzyme form of Npr (pro-Npr). Analysis of the zinc content of purified mutant P57 proteins revealed a dramatic loss of zinc atom as compared with the wild-type P35 protein. In vitro with the aid of exogenous active Npr, the mutant P57 protein could be converted to the mature inactive P35 with an identical NH2-terminal sequence and a molecular mass the same as that of the wild-type P35. From these studies, we conclude that these highly conserved residues (His202, Glu203, His206, and Glu240) are indispensable for zinc binding and protease activity, as well as processing of Npr. In addition, we have clearly demonstrated that maturation of Npr occurs extracellularly via an autocatalytic cleavage of the pro-Npr propeptide. This is the first report of such a maturation mechanism for an extracellular protease in streptomycetes which can serve as a model for further studies on the mechanism of secretion and processing of proteases from Gram-positive bacteria.
authors.affiliation: Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
authors.forename: P C
authors.initials: PC
authors.lastname: Chang
authors.forename: Y H
authors.initials: YH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002413:Cations, Divalent; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D004533:Egtazic Acid; D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc