Abstract
Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC(50) values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants.
Highlights
Our results demonstrate the relative ease at attaining acarbose-resistant cyclodextrin glucanotransferase (CGTase) mutants, increasing the IC50 value for acarbose up to 6,700-fold
Construction and characterization of the purified proteins of the single mutations I61V, D313E, D319E, A230V, K232E, and F283L demonstrated that the A230V, K232E, and F283L mutations were responsible for CGTase insensitivity to acarbose inhibition (Table 1)
IC50 Values of Wild-type and Mutant CGTases—Measurement of -cyclization activity in the presence of varying concentrations of acarbose revealed that mutations at the acceptor subsites of CGTase had a profound effect on inhibitor insensitivity, raising IC50 values by 3,500 – 6,700-fold when compared with wild type (Table 1)
Summary
Cyclodextrin-forming activity in the presence of high acarbose concentrations. Our results demonstrate the relative ease at attaining acarbose-resistant CGTase mutants, increasing the IC50 value for acarbose up to 6,700-fold. Detailed analysis of the insensitive variants highlights the conflicting compromise between native and newly attained enzyme function and subsequent impact on protein stability
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