Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes.
This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.
Sidhu
S S
SS
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Kalmar
G B
GB
Willis
L G
LG
Borgford
T J
TJ
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins; D005075:Biological Evolution; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C092955:protease D, Streptomyces griseus