Structure-based protein engineering is a rational method to improve the catalytic activity and stability of enzymes. A cutinase from Saccharomonospora viridis AHK190, Cut190, hydrolyzes polyethylene terephthalate in the presence of Ca2+. Our crystal structure analysis of the inactive S176A mutant of Cut190 S226P/R228S (Cut190*) showed that Thr107 changes its conformation largely upon binding of Ca2+ and the substrate to Cut190*, and this conformational change facilitates substrate binding. To analyze the role of Thr107, a series of Cut190* mutants, T107A, T107S, T107V, and T107M, were overexpressed in Escherichia coli and purified. The catalytic activities of the mutants were similar to that of Cut190*. Upon mutating to hydrophobic residues, such as Val and Met, the Michaelis-Menten constant increased, possibly due to increased hydrophobic interactions with not only the substrate but also the product. The secondary structure and thermal stability of Cut190*T107 mutants were also analyzed using circular dichroism. Upon mutation, the enzyme structure and stability were almost unchanged. In the presence of Ca2+, unfolded protein states were largely perturbed, similar to what was observed in Cut190*.
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