Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis

Abstract

Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.

Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductase

“…By contrast, the ketone-preferring members of these families often have a tyrosine or phenylalanine residue, but also other residues, as the positional analogue of Trp-23. Considering that enantiopure reduction products of ketones have various applications as chiral building blocks in the organic synthesis of drugs and high-value chemicals, it is quite promising to find significantly improved efficiency for the conversion of an aromatic a-keto ester in a Phe-23 mutant of AKR2B5 (44). We therefore hope that future applications of aldo-keto reductases in biotechnology will benefit from the fundamental studies of structure and function of AKR2B5.…”

“…A characteristic feature of many AKRs is their extremely broad substrate specificity when assayed in vitro (4,44). This unusual promiscuity with respect to the structure of the nonreacting part of the aldehyde or ketone substrate has complicated and made controversial the assignment of a welldefined physiological function, normal or aberrant, to certain AKRs.…”

“…Asn-309 was replaced by Ala or Asp, and a comparison of the crystal structure of Asn-309!Asp bound with NAD þ to the corresponding structure of the wild-type holoenzyme revealed no major structural perturbations caused by the mutation (44). Kinetic parameters have been determined for NADH-dependent reductions of D-galactose and 2-deoxy-Dgalactose catalyzed by wild-type as well as the Ala-309 and Asp-309 mutants.…”

“…The loops 4, 7, C ( Figure 1C) have been shown to be mobile when substrates or inhibitors bind and are thought to confer substrate binding site plasticity which is the likely source of the relaxed kinetic selectivity. AKR2B5 is able to reduce, in addition to xylose, a great variety of aldehydes (R-CHO) whereby R may diverge from an uncharged polyhydroxylated group to hydrophobic aliphatic and aromatic groups harboring various substituents (30,44). To answer the question of how the enzyme brings about the requisite selectivity for its physiological substrate xylose, we have performed a molecular-level analysis of the substrate binding pocket of AKR2B5 (44).…”

“…AKR2B5 is able to reduce, in addition to xylose, a great variety of aldehydes (R-CHO) whereby R may diverge from an uncharged polyhydroxylated group to hydrophobic aliphatic and aromatic groups harboring various substituents (30,44). To answer the question of how the enzyme brings about the requisite selectivity for its physiological substrate xylose, we have performed a molecular-level analysis of the substrate binding pocket of AKR2B5 (44). Scheme 3 shows enzyme-substrate interactions for AKR2B5 which have been predicted by modeling experiments (24).…”