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. 1999 Sep;8(9):1789–1796. doi: 10.1110/ps.8.9.1789

Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2.

G Manco 1, F Febbraio 1, E Adinolfi 1, M Rossi 1
PMCID: PMC2144407  PMID: 10493580

Abstract

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

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Selected References

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  1. Bourne Y., Taylor P., Marchot P. Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex. Cell. 1995 Nov 3;83(3):503–512. doi: 10.1016/0092-8674(95)90128-0. [DOI] [PubMed] [Google Scholar]
  2. Brenner S. The molecular evolution of genes and proteins: a tale of two serines. Nature. 1988 Aug 11;334(6182):528–530. doi: 10.1038/334528a0. [DOI] [PubMed] [Google Scholar]
  3. Brzozowski A. M., Derewenda U., Derewenda Z. S., Dodson G. G., Lawson D. M., Turkenburg J. P., Bjorkling F., Huge-Jensen B., Patkar S. A., Thim L. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature. 1991 Jun 6;351(6326):491–494. doi: 10.1038/351491a0. [DOI] [PubMed] [Google Scholar]
  4. Chothia C., Lesk A. M. The relation between the divergence of sequence and structure in proteins. EMBO J. 1986 Apr;5(4):823–826. doi: 10.1002/j.1460-2075.1986.tb04288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Contreras J. A., Karlsson M., Osterlund T., Laurell H., Svensson A., Holm C. Hormone-sensitive lipase is structurally related to acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. Building of a three-dimensional model for the catalytic domain of hormone-sensitive lipase. J Biol Chem. 1996 Dec 6;271(49):31426–31430. doi: 10.1074/jbc.271.49.31426. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. De Simone G., Manco G., Galdiero S., Lombardi A., Rossi M., Pavone V. Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. Acta Crystallogr D Biol Crystallogr. 1999 Jul;55(Pt 7):1348–1349. doi: 10.1107/s0907444999005156. [DOI] [PubMed] [Google Scholar]
  8. Derewenda U., Brzozowski A. M., Lawson D. M., Derewenda Z. S. Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase. Biochemistry. 1992 Feb 11;31(5):1532–1541. doi: 10.1021/bi00120a034. [DOI] [PubMed] [Google Scholar]
  9. Derewenda U., Swenson L., Green R., Wei Y., Yamaguchi S., Joerger R., Haas M. J., Derewenda Z. S. Current progress in crystallographic studies of new lipases from filamentous fungi. Protein Eng. 1994 Apr;7(4):551–557. doi: 10.1093/protein/7.4.551. [DOI] [PubMed] [Google Scholar]
  10. Feller G., Thiry M., Arpigny J. L., Gerday C. Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144. Gene. 1991 Jun 15;102(1):111–115. doi: 10.1016/0378-1119(91)90548-p. [DOI] [PubMed] [Google Scholar]
  11. Feller G., Thiry M., Gerday C. Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues. DNA Cell Biol. 1991 Jun;10(5):381–388. doi: 10.1089/dna.1991.10.381. [DOI] [PubMed] [Google Scholar]
  12. Hemilä H., Koivula T. T., Palva I. Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: identification of a superfamily of esterases and lipases. Biochim Biophys Acta. 1994 Jan 3;1210(2):249–253. doi: 10.1016/0005-2760(94)90129-5. [DOI] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Kanaya S., Koyanagi T., Kanaya E. An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase. Biochem J. 1998 May 15;332(Pt 1):75–80. doi: 10.1042/bj3320075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krejci E., Duval N., Chatonnet A., Vincens P., Massoulié J. Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6647–6651. doi: 10.1073/pnas.88.15.6647. [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. Langin D., Laurell H., Holst L. S., Belfrage P., Holm C. Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4897–4901. doi: 10.1073/pnas.90.11.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Manco G., Adinolfi E., Pisani F. M., Ottolina G., Carrea G., Rossi M. Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem J. 1998 May 15;332(Pt 1):203–212. doi: 10.1042/bj3320203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martinez C., De Geus P., Lauwereys M., Matthyssens G., Cambillau C. Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature. 1992 Apr 16;356(6370):615–618. doi: 10.1038/356615a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Osterlund T., Contreras J. A., Holm C. Identification of essential aspartic acid and histidine residues of hormone-sensitive lipase: apparent residues of the catalytic triad. FEBS Lett. 1997 Feb 24;403(3):259–262. doi: 10.1016/s0014-5793(97)00063-x. [DOI] [PubMed] [Google Scholar]
  22. Probst M. R., Beer M., Beer D., Jenö P., Meyer U. A., Gasser R. Human liver arylacetamide deacetylase. Molecular cloning of a novel esterase involved in the metabolic activation of arylamine carcinogens with high sequence similarity to hormone-sensitive lipase. J Biol Chem. 1994 Aug 26;269(34):21650–21656. [PubMed] [Google Scholar]
  23. Raibaud A., Zalacain M., Holt T. G., Tizard R., Thompson C. J. Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus. J Bacteriol. 1991 Jul;173(14):4454–4463. doi: 10.1128/jb.173.14.4454-4463.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reddy P. G., Allon R., Mevarech M., Mendelovitz S., Sato Y., Gutnick D. L. Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1. Gene. 1989 Mar 15;76(1):145–152. doi: 10.1016/0378-1119(89)90016-4. [DOI] [PubMed] [Google Scholar]
  25. Sander C., Schneider R. Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins. 1991;9(1):56–68. doi: 10.1002/prot.340090107. [DOI] [PubMed] [Google Scholar]
  26. Sussman J. L., Harel M., Frolow F., Oefner C., Goldman A., Toker L., Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991 Aug 23;253(5022):872–879. doi: 10.1126/science.1678899. [DOI] [PubMed] [Google Scholar]
  27. Wohlleben W., Arnold W., Broer I., Hillemann D., Strauch E., Pühler A. Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene. 1988 Oct 15;70(1):25–37. doi: 10.1016/0378-1119(88)90101-1. [DOI] [PubMed] [Google Scholar]
  28. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]

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