Role of Phe-114 in substrate specificity of Candida tenuis xylose reductase (AKR2B5)

Abstract

The 2.2 Å X-ray crystal structure of Candida tenuis xylose reductase (AKR2B5) bound with NADP⁺ reveals that Phe-114 contributes to the substrate binding pocket of the enzyme. In the related human aldose reductase (AKR1B1), this phenylalanine is replaced by a tryptophan. The side chain of Trp was previously implicated in forming a hydrogen bond with bound substrate or inhibitor. The apparent Michaelis constant of AKR2B5 for xylose (K_m≈90 mM) is 60 times that of AKR1B1, perhaps because critical enzyme–substrate interactions of Trp are not available to Phe-114. We, therefore, prepared a Phe-114→Trp mutant (F114W) of AKR2B5, to mimic the aldose reductase relationship in xylose reductase. Detailed analysis of the kinetic consequences in purified F114W revealed that the K_m values for xylose and xylitol at pH 7.0 and 25°C were increased 5.1- and 4.4-fold, respectively, in the mutant compared with the wild-type. Turnover numbers (k_cat) of F114W for xylose reduction and xylitol oxidation were half those of the wild-type. Apparent dissociation constants of NADH (K_iNADH=44 µM) and NAD⁺ (K_iNAD⁺=177 µM) were increased 1.6- and 1.4-fold in comparison with values of K_iNADH and K_iNAD⁺ for the wild-type, respectively. Catalytic efficiencies (k_cat/K_m) for NADH-dependent reduction of different aldehydes were between 3.1- and 31.5-fold lower than the corresponding k_cat/K_m values of the wild-type. Therefore, replacement of Phe-114 with Trp weakens rather than strengthens apparent substrate binding by AKR2B5, suggesting that xylose reductase exploits residue 114 in a different manner from aldose reductase.

• Aldose reductase
• aldo-keto reductase
• substrate specificity
• xylose reduction