Computational design of Candida boidinii xylose reductase for altered cofactor specificity

Abstract

In this study we introduce a computationally-driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. 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. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR-GGD) being 27-fold more active on NADH. The NADPH-dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10(4)-fold change in specificity to NADH (CbXR-REG). The remaining two variants (CbXR-RTT and CBXR-EQR) had dual cofactor specificity for both nicotinamide cofactors.

Figure 1

(A) The structure of the homology modeled CbXR with NADPH bound and d-xylose situated in a deep cavity inside the (α/ß)₈ barrel. (B) The cofactor binding pocket of wild-type CbXR containing NADH with no hydrogen bonding interactions near the 2′-hydroxyl group. (C) The cofactor binding pocket of CbXR containing hydrogen bonding interactions within 2.5 Å of the 2′-phosphate of NADPH. These hydrogen bonding interactions are important for the specificity of CbXR for NADPH over NADH. This figure was made using PyMOL (Delano Scientific).

Figure 2

Comparison of average hydrophobicity, volume, charge, and BLOSUM62 score for all design positions. Error bars are shown for a 95% confidence interval. No statistically significant signal was found except for charge in position 2, where NADH-preferring residues were found to be more negative than NADPH-preferring residues, which is consistent with previous reports in the literature.

Figure 3

Changes in experimental ground state binding energies from Petschacher et al. versus our calculated changes in interaction energies. Shown are the changes in interaction energy with solvation showing reasonable correlation with the experimental data (R² = 67%), whereas changes in interaction energy without solvation correlated significantly less with the experimental data (R² = 24%) (data not shown).

Figure 4

CbXR-EDS binding pocket containing NADH. The mutated residues Glu-272, Asp-273, and Ser-274 are labeled. Hydrogen bonding interactions are observed within 2.5 Å between the negative Glu-272 and the 3′-OH from NADH. This figure was made using PyMOL (Delano Scientific).

Figure 5

Michaelis-Menten plot for (A) wild type CbXR with NADPH and (B) three tested variants of engineered CbXR with NADH.

Figure 6

Structures of redesigned NAD(P)H binding pockets. (A) CbXR-GGD and (B) CbXR-MGD establish new hydrogen bond interactions between the mutated residues in CbXR and the bridging phosphates in NADH. The net charge change of these mutations is negative which may serve to compensate for the lack of negative 2′-phosphate in NADH. The mutations to glycine may serve to add conformational flexibility in the backbone to allow proper positioning of the NADH. CbXR-RTT, the mutation predicted by IPRO that was experimentally found to have dual cofactor specificity, bound to NADH (C) and NADPH (D). New hydrogen bond interactions are shown stabilizing the 3′-phosphate in NADH and NADPH from Arg-272, which may be the cause of the dual cofactor specificity. In NADPH, new hydrogen bonds are found to stabilize the 2′-phosphate group from Arg-272 and Thr-274. A neutral net change in charge is thought to contribute to dual cofactor specificity as well. All hydrogen bonds shown are within 2.5 Å. This figure was made using PyMOL (Delano Scientific).

Figure 7

Plots of the natural log of specific activity toward NADPH (A) or NADH (B) versus interaction energy for CbXR mutants described in this study. The correlation coefficient for mutants yielding activity for NADPH is 79%, whereas the correlation is only 30% for NADH.