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. 1999 Oct;8(10):1990–2000. doi: 10.1110/ps.8.10.1990

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

J Zuegg 1, K Gruber 1, M Gugganig 1, U G Wagner 1, C Kratky 1
PMCID: PMC2144128  PMID: 10548044

Abstract

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.

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Selected References

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  1. Barth A., Frost K., Wahab M., Brandt W., Schadler H. D., Franke R. Classification of serine proteases derived from steric comparisons of their active sites, part II: "Ser, His, Asp arrangements in proteolytic and nonproteolytic proteins". Drug Des Discov. 1994 Nov;12(2):89–111. [PubMed] [Google Scholar]
  2. Bauer M, Griengl H, Steiner W. Kinetic studies on the enzyme (S)-hydroxynitrile lyase from hevea brasiliensis using initial rate methods and progress curve analysis . Biotechnol Bioeng. 1999 Jan 5;62(1):20–29. doi: 10.1002/(sici)1097-0290(19990105)62:1<20::aid-bit3>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
  3. Cygler M., Schrag J. D., Sussman J. L., Harel M., Silman I., Gentry M. K., Doctor B. P. Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci. 1993 Mar;2(3):366–382. doi: 10.1002/pro.5560020309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Grochulski P., Li Y., Schrag J. D., Bouthillier F., Smith P., Harrison D., Rubin B., Cygler M. Insights into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem. 1993 Jun 15;268(17):12843–12847. [PubMed] [Google Scholar]
  5. Grochulski P., Li Y., Schrag J. D., Cygler M. Two conformational states of Candida rugosa lipase. Protein Sci. 1994 Jan;3(1):82–91. doi: 10.1002/pro.5560030111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gruber K., Klintschar G., Hayn M., Schlacher A., Steiner W., Kratky C. Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry. 1998 Sep 29;37(39):13475–13485. doi: 10.1021/bi980864l. [DOI] [PubMed] [Google Scholar]
  7. Hasslacher M., Kratky C., Griengl H., Schwab H., Kohlwein S. D. Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis. Proteins. 1997 Mar;27(3):438–449. [PubMed] [Google Scholar]
  8. Hasslacher M., Schall M., Hayn M., Bona R., Rumbold K., Lückl J., Griengl H., Kohlwein S. D., Schwab H. High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expr Purif. 1997 Oct;11(1):61–71. doi: 10.1006/prep.1997.0765. [DOI] [PubMed] [Google Scholar]
  9. Hasslacher M., Schall M., Hayn M., Griengl H., Kohlwein S. D., Schwab H. (S)-hydroxynitrile lyase from Hevea brasiliensis. Ann N Y Acad Sci. 1996 Oct 12;799:707–712. doi: 10.1111/j.1749-6632.1996.tb33278.x. [DOI] [PubMed] [Google Scholar]
  10. Hasslacher M., Schall M., Hayn M., Griengl H., Kohlwein S. D., Schwab H. Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue. J Biol Chem. 1996 Mar 8;271(10):5884–5891. doi: 10.1074/jbc.271.10.5884. [DOI] [PubMed] [Google Scholar]
  11. Hughes J., Carvalho F. J., Hughes M. A. Purification, characterization, and cloning of alpha-hydroxynitrile lyase from cassava (Manihot esculenta Crantz). Arch Biochem Biophys. 1994 Jun;311(2):496–502. doi: 10.1006/abbi.1994.1267. [DOI] [PubMed] [Google Scholar]
  12. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  13. Kuroki G. W., Conn E. E. Mandelonitrile lyase from Ximenia americana L.: stereospecificity and lack of flavin prosthetic group. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6978–6981. doi: 10.1073/pnas.86.18.6978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ménard R., Khouri H. E., Plouffe C., Dupras R., Ripoll D., Vernet T., Tessier D. C., Lalberté F., Thomas D. Y., Storer A. C. A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain. Biochemistry. 1990 Jul 17;29(28):6706–6713. doi: 10.1021/bi00480a021. [DOI] [PubMed] [Google Scholar]
  15. Ollis D. L., Cheah E., Cygler M., Dijkstra B., Frolow F., Franken S. M., Harel M., Remington S. J., Silman I., Schrag J. The alpha/beta hydrolase fold. Protein Eng. 1992 Apr;5(3):197–211. doi: 10.1093/protein/5.3.197. [DOI] [PubMed] [Google Scholar]
  16. Schmidt A., Gübitz G. M., Kratky C. Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry. 1999 Feb 23;38(8):2403–2412. doi: 10.1021/bi982108l. [DOI] [PubMed] [Google Scholar]
  17. Schmidt A., Schlacher A., Steiner W., Schwab H., Kratky C. Structure of the xylanase from Penicillium simplicissimum. Protein Sci. 1998 Oct;7(10):2081–2088. doi: 10.1002/pro.5560071004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schrag J. D., Cygler M. 1.8 A refined structure of the lipase from Geotrichum candidum. J Mol Biol. 1993 Mar 20;230(2):575–591. doi: 10.1006/jmbi.1993.1171. [DOI] [PubMed] [Google Scholar]
  19. Wagner U. G., Hasslacher M., Griengl H., Schwab H., Kratky C. Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis. Structure. 1996 Jul 15;4(7):811–822. doi: 10.1016/s0969-2126(96)00088-3. [DOI] [PubMed] [Google Scholar]
  20. Wajant H., Effenberger F. Hydroxynitrile lyases of higher plants. Biol Chem. 1996 Oct;377(10):611–617. [PubMed] [Google Scholar]
  21. Wajant H., Pfizenmaier K. Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis. J Biol Chem. 1996 Oct 18;271(42):25830–25834. doi: 10.1074/jbc.271.42.25830. [DOI] [PubMed] [Google Scholar]
  22. Wallace A. C., Laskowski R. A., Thornton J. M. Derivation of 3D coordinate templates for searching structural databases: application to Ser-His-Asp catalytic triads in the serine proteinases and lipases. Protein Sci. 1996 Jun;5(6):1001–1013. doi: 10.1002/pro.5560050603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Xu L. L., Singh B. K., Conn E. E. Purification and characterization of acetone cyanohydrin lyase from Linum usitatissimum. Arch Biochem Biophys. 1988 Jun;263(2):256–263. doi: 10.1016/0003-9861(88)90634-0. [DOI] [PubMed] [Google Scholar]

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