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Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.
Biochem J. 2006 Jan 01; 393(Pt 1):51-8.BJ

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.

Authors+Show Affiliations

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16336198

Citation

Kratzer, Regina, et al. "Probing the Substrate Binding Site of Candida Tenuis Xylose Reductase (AKR2B5) With Site-directed Mutagenesis." The Biochemical Journal, vol. 393, no. Pt 1, 2006, pp. 51-8.
Kratzer R, Leitgeb S, Wilson DK, et al. Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. Biochem J. 2006;393(Pt 1):51-8.
Kratzer, R., Leitgeb, S., Wilson, D. K., & Nidetzky, B. (2006). Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. The Biochemical Journal, 393(Pt 1), 51-8.
Kratzer R, et al. Probing the Substrate Binding Site of Candida Tenuis Xylose Reductase (AKR2B5) With Site-directed Mutagenesis. Biochem J. 2006 Jan 1;393(Pt 1):51-8. PubMed PMID: 16336198.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. AU - Kratzer,Regina, AU - Leitgeb,Stefan, AU - Wilson,David K, AU - Nidetzky,Bernd, PY - 2005/12/13/pubmed PY - 2006/3/17/medline PY - 2005/12/13/entrez SP - 51 EP - 8 JF - The Biochemical journal JO - Biochem. J. VL - 393 IS - Pt 1 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. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/16336198/Probing_the_substrate_binding_site_of_Candida_tenuis_xylose_reductase__AKR2B5__with_site_directed_mutagenesis_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20050831 DB - PRIME DP - Unbound Medicine ER -