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Bioengineering coagulation factor Xa substrate specificity into Streptomyces griseus trypsin

Extended substrate specificity is exhibited by a number of highly evolved members of the SI peptidase family, such as the vertebrate blood coagulation proteases. Dissection of this substrate specificity has been hindered by the complexity and physiological requirements of these proteases. In order to understand the mechanisms of extended substrate specificity, a bacterial trypsin-like enzyme, Streptomyces griseus trypsin (SGT), was chosen as a scaffold for the introduction of extended substrate specificity through structure-based genetic engineering. Recombinant and mutant SGT proteases were produced in a B. subtilis expression system, which constitutively secretes active protease into the extracellular medium at greater than 15 mg/L of culture. Comparison of the recombinant wild-type protease to the natively produced enzyme demonstrated near identity in enzymatic and structural properties. To begin construction of a high specificity protease, four mutants in the S1 substrate binding pocket (T190A, T190S, T190V, and T190P) were produced and examined for differences in the Arg:Lys preference. Only the T190P mutant of SGT demonstrated a significant increase in PI arginine to lysine preference a three-fold improvement to 16:1 with only a minor reduction in catalytic activity (kcat reduction of 25%). The 1.9 A resolution crystal structure of T190P mutant of SGT in complex with the small molecule inhibitor benzamidine was subsequently determined. The model shows that the increased preference for Arg over Lys side chains in the SI pocket is the result of the second shell residues of the SI pocket, particularly by the N-terminal residue of the protease which does not conflict with the introduced proline ring.