Natural and artificial evolution in the serine proteases of Streptomyces griseus

The natural diversity of the Streptomyces griseus serine protease family was investigated; the insights gained were used to design enzymes with altered specificities. Genes encoding S. griseus protease E and two previously uncharacterized proteases (S. griseus proteases C and D, SGPC and SGPD) were isolated and sequenced. A shuttle vector was constructed to allow for genetic manipulations in Escherichia coli and protein secretion in Bacillus subtilis; the recombinant gene products were purified and characterized. The two newly characterized enzymes possessed domains unique among bacterial proteases: SGPC contained a carboxyl-terminal domain homologous to chitin binding domains while SGPD possessed an unusually long amino-terminal prepeptide with characteristics of mitochondria1 import signals. Furthermore, recombinant SGPD existed as a very stable homodimer. The S. griseus proteases are secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity. S. griseus protease B (SGPB, a previously characterized enzyme) has primary specificity for large hydrophobic substrates. We genetically substituted the Leu residue at the promature junction PI site of SGPB with various amino acids; substitution with poor SGPB substrates reduced or abolished active protease secretion in E. coli. Thus, it was demonstrated that the specificity of SGPB is constrained by the promature junction sequence; active protease production is dependent on the efficiency of autocatalytic processing. The link between substrate specificity and proteolytic activity was used to select enzymes with altered primary specificities: loss of proteolytic activity due to mutations at the promature junction PI site could be restored by compensatory changes in enzyme specificity. Sequence alignments and crystallographic data were used to design an E. coli expression library containing 29,952 possible SGPB mutants with variations at seven sites involved in conferring primary specificity. A visual screening strategy allowing for the rapid analysis of up to 106 individual variants was developed to detect active protease secretion. The expression library was screened in conjunction with various promature junction sequences; variants producing increased proteolytic activity were isolated. Variant enzymes with increased specificity towards various hydrophobic and polar substrates were isolated.