PROTEASE EVOLUTION IN STREPTOMYCES-GRISEUS - DISCOVERY OF A NOVEL DIMERIC ENZYME

This report describes the cloning and sequencing of a novel protease g ene derived from Streptomyces griseus, Also described is the heterolog ous expression of the gene in Bacillus subtilis and characterization o f the gene product, The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A sign ificant component of the enzyme preregion was found to be homologous w ith the mitochondrial import signal of hsp60. The sprD gene was subclo ned into an Escherichia coli/B. subtilis shuttle vector system such th at the pro mature portion of SGPD was fused in flame with the promoter , ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active pro tease was purified, SGPD has a high degree of sequence homology to pre viously described S. griseus proteases A, B, C, and E and the alpha-ly tic protease of Lysobacter enzymogenes, but unlike all previously char acterized members of the chymotrypsin superfamily, the recombinant SGP D forms a stable alpha(2) dimer. The amino acid sequence of the protei n in the region of the specificity pocket is similar to that of S. gri seus proteases A, B, and C, The purified enzyme was found to have a pr imary specificity for large aliphatic or aromatic amino acids. Nucleot ide sequence data were used to construct a phylogenetic tree using a m ethod of maximum parsimony which reflects the relationships and potent ially the lineage of the chymotrypsin-like proteases of S. griseus.