title: Generation of a thermostable and denaturant-resistant peptide ligase.
abstract: The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Koman
authors.initials: K
authors.lastname: Joe
authors.forename: Thor J
authors.initials: TJ
authors.lastname: Borgford
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology; D013696:Temperature
chemical_list: D008025:Ligases