Engineering the primary substrate specificity of Streptomyces griseus trypsin.
Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures. Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed. The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site. This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%). The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A). The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets.
Page
Michael J
MJ
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Wong
Sui-Lam
SL
Hewitt
Jeff
J
Strynadka
Natalie C J
NC
MacGillivray
Ross T A
RT
D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011402:Pronase / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D014357:Trypsin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D014357:Trypsin; D011402:Pronase; D002118:Calcium