(156e) Engineering the Cofactor Specificity of Candida Boidinii Xylose Reductase

In this study we introduce a computationally-driven enzyme redesign workflow for altered cofactor specificity from NADPH to NADH. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the Iterative Protein Redesign and Optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non-native cofactor NADH. Using the modified IPRO, we identified ten variants that improve the computationally assessed binding energy with NADH by introducing mutations in the CbXR binding pocket. We also computationally assessed the effects of the predicted mutations on the retention or abolishment of affinity with the native cofactor NADPH. Site directed mutagenesis revealed that seven out of ten mutants possessed significant xylose reductase activity utilizing NADH. The NADPH-dependent activity of eight out of ten predicted designs was either completely abolished or significantly diminished by 90% or greater. The remaining two variants (CbXR-RTT and CBXR-EQR) were found to have dual cofactor specificity for both nicotinamide cofactors. Both of these trends were in agreement with our computational predictions assessing the effects of the mutations on binding energies. Finally, we compare the effects of the mutations on the binding of both cofactors with the wild-type using molecular dynamics simulations, and perform a Shannon entropic analysis on the sequence diversity of the aldo-keto reductase superfamily combined with knowledge of the structure of the CbXR binding pocket in order to elucidate further positions for design to increase NADH affinity.