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

@article{citeulike:2298340, 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.}, address = {Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.}, author = {Kratzer, R. and Leitgeb, S. and Wilson, D. K. and Nidetzky, B.}, citeulike-article-id = {2298340}, citeulike-linkout-0 = {http://dx.doi.org/10.1042/BJ20050831}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/16336198}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=16336198}, day = {1}, doi = {10.1042/BJ20050831}, issn = {1470-8728}, journal = {Biochem J}, keywords = {modeling, mutagenesis}, month = {January}, number = {Pt 1}, pages = {51--58}, posted-at = {2008-01-28 16:52:37}, priority = {2}, title = {Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.}, url = {http://dx.doi.org/10.1042/BJ20050831}, volume = {393}, year = {2006} }

TY - JOUR ID - citeulike:2298340 L3 - citeulike-article-id:2298340 N2 - 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. IS - Pt 1 JF - Biochem J SN - 1470-8728 AD - Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. EP - 58 TI - Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. VL - 393 SP - 51 KW - modeling KW - mutagenesis AU - Kratzer, R AU - Leitgeb, S AU - Wilson, DK AU - Nidetzky, B PY - 2006/01/01/ UR - http://dx.doi.org/10.1042/BJ20050831 ER -