The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

Abstract

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…' Both postulated options have, to a certain extend, been realized since then. Petschacher et al (2005) were able to shift the cofactor preference of the xylose reductase of Candida tenuis from NADPH towards NADH by site-directed mutagenesis. Regrettably, this paper does not mention the xylose fermentation properties of the strains expressing the engineered enzyme.…”

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

“…27,53,72 We defined Positions 1-3 as the residues that are aligned to K274, S275, and N276, respectively. Positions 1-3 are nearby the phosphate group in NADPH, but are over 12Å from the hydride transfer site in the catalytic mechanism, highlighting that these positions affect cofactor specificity and affinity but are not directly involved in the reaction.…”

“…To test this, we contrasted calculated interaction energy values (through CHARMM 75,76 ) with published kinetic parameter data from a study aimed at changing specificity from NADPH to NADH in CtXR. 27 We compare the results of interaction energy changes calculated with and without solvation effects to determine whether the substantially increased computational cost needed for solvation is necessary. The crystal structure of CtXR with NADH bound (PDB:1MI3) provided the starting coordinates for this analysis.…”

“…55 This enzyme selectively binds NADPH over NADH by roughly 20-fold. 27 In contrast, CbXR (62% homologous to CtXR) is strictly an NADPH-dependent enzyme. The structure of the homology modeled CbXR is shown in Figure 1(A), with NADPH bound and D-xylose situated inside the (a/ß) 8 barrel.…”

“…Site-directed mutagenesisbased studies also successfully pinpointed sets of mutations leading to complete reversal of Candida tenuis xylose reductase (CtXR) cofactor specificity from NADPH to NADH (reduced form of NAD þ ). 26,27 Similarly, Liang et al 28 used a semirational approach called combinatorial active site saturation (CASTing) to switch cofactor preference from NADPH to NADH in PsXR.…”