Engineering the Primary Substrate Specificity of Streptomyces griseus Trypsin

Abstract

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.

Cloning and expression of alkaline protease genes from two salt-tolerant alkaliphilic actinomycetes in E. coli

“…Page and coworkers [9] chose Streptomyces griseus trypsin (SGT) 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.…”

Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na+-activated Trypsin

“…Sodium ions are present at full occupancy with temperature factors of 17.54 Å 2 and 20.47 Å 2 in molecules A and B, respectively. observed with wild-type SGT, 25 yet sufficient for purification to homogeneity followed by kinetic and structural analyses. Protein purity was N98% by active site titration using PPACK.…”

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

“…; 12; 13 In this system, SGT and its mutants are constitutively expressed in their active form and secreted directly into the culture supernatant. Combinatorial enzyme design was performed using purified plasmid DNA obtained from recombinant clones using the QIAprep Miniprep Spin kit (Qiagen, Valencia, CA).…”

“…For the example protease pair used in this study, the core of both human coagulation factor Xa (FXa) and the bacterial Streptomyces griseus trypsin (SGT) are highly similar (Figure 1B). 11; 12; 13 However, the surface differences between these two proteins are sufficiently extensive to warrant the use of a color-coded pie diagram as a notation system rather than writing out the 54 individual point mutations (Figure 1C). The considerable evolutionary distance between these two proteins are only one problem amongst many for interconverting their properties.…”

“…We have been engaged in converting a bacterial trypsin into an artificial blood coagulation factor 11; 12; 13 and hindered by many of these difficulties that limit throughput of any design strategy. In particular, trypsin-like proteases are rarely found in bacterial genomes and not readily expressed in soluble form by bacteria 4.…”