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. 2003 Jul 15;373(Pt 2):319–326. doi: 10.1042/BJ20030286

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

Kathryn L Kavanagh 1, Mario Klimacek 1, Bernd Nidetzky 1, David K Wilson 1
PMCID: PMC1223518  PMID: 12733986

Abstract

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

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Selected References

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  1. Banta Scott, Swanson Barbara A., Wu Shan, Jarnagin Alisha, Anderson Stephen. Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A. Protein Eng. 2002 Feb;15(2):131–140. doi: 10.1093/protein/15.2.131. [DOI] [PubMed] [Google Scholar]
  2. Banta Scott, Swanson Barbara A., Wu Shan, Jarnagin Alisha, Anderson Stephen. Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis. Biochemistry. 2002 May 21;41(20):6226–6236. doi: 10.1021/bi015987b. [DOI] [PubMed] [Google Scholar]
  3. Bennett M. J., Schlegel B. P., Jez J. M., Penning T. M., Lewis M. Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. Biochemistry. 1996 Aug 20;35(33):10702–10711. doi: 10.1021/bi9604688. [DOI] [PubMed] [Google Scholar]
  4. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  5. Carugo O., Argos P. NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding. Proteins. 1997 May;28(1):10–28. doi: 10.1002/(sici)1097-0134(199705)28:1<10::aid-prot2>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]
  6. Ellis E. M., Hayes J. D. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase. Biochem J. 1995 Dec 1;312(Pt 2):535–541. doi: 10.1042/bj3120535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esnouf R. M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J Mol Graph Model. 1997 Apr;15(2):132-4, 112-3. doi: 10.1016/S1093-3263(97)00021-1. [DOI] [PubMed] [Google Scholar]
  8. Granström T., Leisola M. Controlled transient changes reveal differences in metabolite production in two Candida yeasts. Appl Microbiol Biotechnol. 2002 Feb 1;58(4):511–516. doi: 10.1007/s00253-001-0921-4. [DOI] [PubMed] [Google Scholar]
  9. Hahn-Hägerdal B., Wahlbom C. F., Gárdonyi M., van Zyl W. H., Cordero Otero R. R., Jönsson L. J. Metabolic engineering of Saccharomyces cerevisiae for xylose utilization. Adv Biochem Eng Biotechnol. 2001;73:53–84. doi: 10.1007/3-540-45300-8_4. [DOI] [PubMed] [Google Scholar]
  10. Hur E., Wilson D. K. Crystallization and aldo-keto reductase activity of Gcy1p from Saccharomyces cerevisiae. Acta Crystallogr D Biol Crystallogr. 2000 Jun;56(Pt 6):763–765. doi: 10.1107/s0907444900004704. [DOI] [PubMed] [Google Scholar]
  11. Hur E., Wilson D. K. The crystal structure of the GCY1 protein from S. cerevisiae suggests a divergent aldo-keto reductase catalytic mechanism. Chem Biol Interact. 2001 Jan 30;130-132(1-3):527–536. doi: 10.1016/s0009-2797(00)00296-9. [DOI] [PubMed] [Google Scholar]
  12. Häcker B., Habenicht A., Kiess M., Mattes R. Xylose utilisation: cloning and characterisation of the Xylose reductase from Candida tenuis. Biol Chem. 1999 Dec;380(12):1395–1403. doi: 10.1515/BC.1999.179. [DOI] [PubMed] [Google Scholar]
  13. Jez J. M., Penning T. M. The aldo-keto reductase (AKR) superfamily: an update. Chem Biol Interact. 2001 Jan 30;130-132(1-3):499–525. doi: 10.1016/s0009-2797(00)00295-7. [DOI] [PubMed] [Google Scholar]
  14. Kador P. F., Robison W. G., Jr, Kinoshita J. H. The pharmacology of aldose reductase inhibitors. Annu Rev Pharmacol Toxicol. 1985;25:691–714. doi: 10.1146/annurev.pa.25.040185.003355. [DOI] [PubMed] [Google Scholar]
  15. Kavanagh Kathryn L., Klimacek Mario, Nidetzky Bernd, Wilson David K. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis. Biochemistry. 2002 Jul 16;41(28):8785–8795. doi: 10.1021/bi025786n. [DOI] [PubMed] [Google Scholar]
  16. Kozma Evelin, Brown Elaine, Ellis Elizabeth M., Lapthorn Adrian J. The crystal structure of rat liver AKR7A1. A dimeric member of the aldo-keto reductase superfamily. J Biol Chem. 2002 Feb 11;277(18):16285–16293. doi: 10.1074/jbc.M110808200. [DOI] [PubMed] [Google Scholar]
  17. Kubiseski T. J., Flynn T. G. Studies on human aldose reductase. Probing the role of arginine 268 by site-directed mutagenesis. J Biol Chem. 1995 Jul 14;270(28):16911–16917. [PubMed] [Google Scholar]
  18. Lee H. The structure and function of yeast xylose (aldose) reductases. Yeast. 1998 Aug;14(11):977–984. doi: 10.1002/(SICI)1097-0061(199808)14:11<977::AID-YEA302>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  19. Lunzer R., Mamnun Y., Haltrich D., Kulbe K. D., Nidetzky B. Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases. Biochem J. 1998 Nov 15;336(Pt 1):91–99. doi: 10.1042/bj3360091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matsuura K., Tamada Y., Sato K., Iwasa H., Miwa G., Deyashiki Y., Hara A. Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3 alpha-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis. Biochem J. 1997 Feb 15;322(Pt 1):89–93. doi: 10.1042/bj3220089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mayr P., Brüggler K., Kulbe K. D., Nidetzky B. D-Xylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with different coenzyme specificities. J Chromatogr B Biomed Sci Appl. 2000 Jan 14;737(1-2):195–202. doi: 10.1016/s0378-4347(99)00380-1. [DOI] [PubMed] [Google Scholar]
  22. Miller J. V., Estell D. A., Lazarus R. A. Purification and characterization of 2,5-diketo-D-gluconate reductase from Corynebacterium sp. J Biol Chem. 1987 Jul 5;262(19):9016–9020. [PubMed] [Google Scholar]
  23. Neuhauser W., Haltrich D., Kulbe K. D., Nidetzky B. NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme. Biochem J. 1997 Sep 15;326(Pt 3):683–692. doi: 10.1042/bj3260683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Neuhauser W., Haltrich D., Kulbe K. D., Nidetzky B. Noncovalent enzyme-substrate interactions in the catalytic mechanism of yeast aldose reductase. Biochemistry. 1998 Jan 27;37(4):1116–1123. doi: 10.1021/bi9717800. [DOI] [PubMed] [Google Scholar]
  25. Nidetzky B., Mayr P., Neuhauser W., Puchberger M. Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis. Chem Biol Interact. 2001 Jan 30;130-132(1-3):583–595. doi: 10.1016/s0009-2797(00)00285-4. [DOI] [PubMed] [Google Scholar]
  26. Ostergaard S., Olsson L., Nielsen J. Metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2000 Mar;64(1):34–50. doi: 10.1128/mmbr.64.1.34-50.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ratnam K., Ma H., Penning T. M. The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase. Biochemistry. 1999 Jun 15;38(24):7856–7864. doi: 10.1021/bi982838t. [DOI] [PubMed] [Google Scholar]
  28. Rees-Milton K. J., Jia Z., Green N. C., Bhatia M., El-Kabbani O., Flynn T. G. Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities. Arch Biochem Biophys. 1998 Jul 15;355(2):137–144. doi: 10.1006/abbi.1998.0721. [DOI] [PubMed] [Google Scholar]
  29. Sanli G., Blaber M. Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor. J Mol Biol. 2001 Jun 22;309(5):1209–1218. doi: 10.1006/jmbi.2001.4739. [DOI] [PubMed] [Google Scholar]
  30. Senac T., Hahn-Hägerdal B. Effects of increased transaldolase activity on D-xylulose and D-glucose metabolism in Saccharomyces cerevisiae cell extracts. Appl Environ Microbiol. 1991 Jun;57(6):1701–1706. doi: 10.1128/aem.57.6.1701-1706.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Verduyn C., Van Kleef R., Frank J., Schreuder H., Van Dijken J. P., Scheffers W. A. Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis. Biochem J. 1985 Mar 15;226(3):669–677. doi: 10.1042/bj2260669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wilson D. K., Bohren K. M., Gabbay K. H., Quiocho F. A. An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science. 1992 Jul 3;257(5066):81–84. doi: 10.1126/science.1621098. [DOI] [PubMed] [Google Scholar]

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