Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):61–64. doi: 10.1042/bj3230061

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

K Matsuura 1, Y Deyashiki 1, K Sato 1, N Ishida 1, G Miwa 1, A Hara 1
PMCID: PMC1218315  PMID: 9173902

Abstract

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

Full Text

The Full Text of this article is available as a PDF (181.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barski O. A., Gabbay K. H., Grimshaw C. E., Bohren K. M. Mechanism of human aldehyde reductase: characterization of the active site pocket. Biochemistry. 1995 Sep 5;34(35):11264–11275. doi: 10.1021/bi00035a036. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Binstock J. M., Iyer R. B., Hamby C. V., Fried V. A., Schwartz I. S., Weinstein B. I., Southren A. L. Human hepatic 3 alpha-hydroxysteroid dehydrogenase: possible identity with human hepatic chlordecone reductase. Biochem Biophys Res Commun. 1992 Sep 16;187(2):760–766. doi: 10.1016/0006-291x(92)91260-w. [DOI] [PubMed] [Google Scholar]
  4. Bohren K. M., Bullock B., Wermuth B., Gabbay K. H. The aldo-keto reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases. J Biol Chem. 1989 Jun 5;264(16):9547–9551. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Bruce N. C., Willey D. L., Coulson A. F., Jeffery J. Bacterial morphine dehydrogenase further defines a distinct superfamily of oxidoreductases with diverse functional activities. Biochem J. 1994 May 1;299(Pt 3):805–811. doi: 10.1042/bj2990805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ciaccio P. J., Jaiswal A. K., Tew K. D. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics. J Biol Chem. 1994 Jun 3;269(22):15558–15562. [PubMed] [Google Scholar]
  8. Deyashiki Y., Ogasawara A., Nakayama T., Nakanishi M., Miyabe Y., Sato K., Hara A. Molecular cloning of two human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder. Biochem J. 1994 Apr 15;299(Pt 2):545–552. doi: 10.1042/bj2990545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deyashiki Y., Tamada Y., Miyabe Y., Nakanishi M., Matsuura K., Hara A. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver. J Biochem. 1995 Aug;118(2):285–290. doi: 10.1093/oxfordjournals.jbchem.a124904. [DOI] [PubMed] [Google Scholar]
  10. Deyashiki Y., Taniguchi H., Amano T., Nakayama T., Hara A., Sawada H. Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 alpha-hydroxysteroid dehydrogenase activity. Biochem J. 1992 Mar 15;282(Pt 3):741–746. doi: 10.1042/bj2820741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehrig T., Bohren K. M., Prendergast F. G., Gabbay K. H. Mechanism of aldose reductase inhibition: binding of NADP+/NADPH and alrestatin-like inhibitors. Biochemistry. 1994 Jun 14;33(23):7157–7165. doi: 10.1021/bi00189a019. [DOI] [PubMed] [Google Scholar]
  12. Hara A., Matsuura K., Tamada Y., Sato K., Miyabe Y., Deyashiki Y., Ishida N. Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells. Biochem J. 1996 Jan 15;313(Pt 2):373–376. doi: 10.1042/bj3130373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hara A., Taniguchi H., Nakayama T., Sawada H. Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver. J Biochem. 1990 Aug;108(2):250–254. doi: 10.1093/oxfordjournals.jbchem.a123189. [DOI] [PubMed] [Google Scholar]
  14. Harrison D. H., Bohren K. M., Ringe D., Petsko G. A., Gabbay K. H. An anion binding site in human aldose reductase: mechanistic implications for the binding of citrate, cacodylate, and glucose 6-phosphate. Biochemistry. 1994 Mar 1;33(8):2011–2020. doi: 10.1021/bi00174a006. [DOI] [PubMed] [Google Scholar]
  15. Hoog S. S., Pawlowski J. E., Alzari P. M., Penning T. M., Lewis M. Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2517–2521. doi: 10.1073/pnas.91.7.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Ohara H., Miyabe Y., Deyashiki Y., Matsuura K., Hara A. Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver. Biochem Pharmacol. 1995 Jul 17;50(2):221–227. doi: 10.1016/0006-2952(95)00124-i. [DOI] [PubMed] [Google Scholar]
  18. Ohara H., Nakayama T., Deyashiki Y., Hara A., Miyabe Y., Tsukada F. Reduction of prostaglandin D2 to 9 alpha,11 beta-prostaglandin F2 by a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme. Biochim Biophys Acta. 1994 Nov 17;1215(1-2):59–65. doi: 10.1016/0005-2760(94)90091-4. [DOI] [PubMed] [Google Scholar]
  19. Penning T. M., Smithgall T. E., Askonas L. J., Sharp R. B. Rat liver 3 alpha-hydroxysteroid dehydrogenase. Steroids. 1986 Apr-May;47(4-5):221–247. doi: 10.1016/0039-128x(86)90094-2. [DOI] [PubMed] [Google Scholar]
  20. Stolz A., Hammond L., Lou H., Takikawa H., Ronk M., Shively J. E. cDNA cloning and expression of the human hepatic bile acid-binding protein. A member of the monomeric reductase gene family. J Biol Chem. 1993 May 15;268(14):10448–10457. [PubMed] [Google Scholar]
  21. Stolz A., Takikawa H., Sugiyama Y., Kuhlenkamp J., Kaplowitz N. 3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance. J Clin Invest. 1987 Feb;79(2):427–434. doi: 10.1172/JCI112829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tarle I., Borhani D. W., Wilson D. K., Quiocho F. A., Petrash J. M. Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110. J Biol Chem. 1993 Dec 5;268(34):25687–25693. [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Wilson D. K., Nakano T., Petrash J. M., Quiocho F. A. 1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor. Biochemistry. 1995 Nov 7;34(44):14323–14330. doi: 10.1021/bi00044a009. [DOI] [PubMed] [Google Scholar]
  26. Wilson D. K., Tarle I., Petrash J. M., Quiocho F. A. Refined 1.8 A structure of human aldose reductase complexed with the potent inhibitor zopolrestat. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9847–9851. doi: 10.1073/pnas.90.21.9847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Winters C. J., Molowa D. T., Guzelian P. S. Isolation and characterization of cloned cDNAs encoding human liver chlordecone reductase. Biochemistry. 1990 Jan 30;29(4):1080–1087. doi: 10.1021/bi00456a034. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES