Active-site variants of Streptomyces griseus protease B with peptide-ligation activity

Background: Peptide-ligating technologies facilitate a range of manipulatio ns for the study of protein structure and function that are not possible us ing conventional genetic or mutagenic methods. To different extents, the cu rrently available enzymatic and nonenzymatic methodologies are syntheticall y demanding, sequence-dependent and/or sensitive to denaturants. No single coupling method is universally applicable, Accordingly, new strategies for peptide ligation are sought. Results: Site-specific Variants (Ser195-->Gly, S195G, and Ser195-->Ala, S19 5A) of Streptomyces griseus protease B (SGPB) were generated that efficient ly catalyze peptide ligation (i.e., aminolysis of ester-, thioester- and pa ra-nitroanilide-activated peptides). The variants also showed reduced hydro lytic activity relative to the wild-type enzyme. The ratio of aminolysis to hydrolysis was greater for the S195A variant, which was also capable of ca talyzing ligation in concentrations of urea as high as 2 M. Conclusions: Mutagenic substitution of the active-site serine residue of SG PB by either glycine or alanine has created a unique class of peptide-ligat ing catalysts that are useful for coupling relatively stable ester- and par a- nitroanilide-activated substrates. Ligation proceeds through an acyl-enz yme intermediate involving His57. Serine to alanine mutations may provide a general strategy for converting proteases with chymotrypsin-like protein f olds into peptide-coupling enzymes.