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. 1998 May 15;332(Pt 1):203–212. doi: 10.1042/bj3320203

Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily.

G Manco 1, E Adinolfi 1, F M Pisani 1, G Ottolina 1, G Carrea 1, M Rossi 1
PMCID: PMC1219469  PMID: 9576869

Abstract

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

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Selected References

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  1. Aguilar C. F., Sanderson I., Moracci M., Ciaramella M., Nucci R., Rossi M., Pearl L. H. Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability. J Mol Biol. 1997 Sep 5;271(5):789–802. doi: 10.1006/jmbi.1997.1215. [DOI] [PubMed] [Google Scholar]
  2. Alon R. N., Gutnick D. L. Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: sequence analysis and over-expression in Escherichia coli. FEMS Microbiol Lett. 1993 Sep 15;112(3):275–280. doi: 10.1111/j.1574-6968.1993.tb06462.x. [DOI] [PubMed] [Google Scholar]
  3. Brown W. C., Campbell J. L. A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli. Gene. 1993 May 15;127(1):99–103. doi: 10.1016/0378-1119(93)90622-a. [DOI] [PubMed] [Google Scholar]
  4. Ehnholm C., Kuusi T. Preparation, characterization, and measurement of hepatic lipase. Methods Enzymol. 1986;129:716–738. doi: 10.1016/0076-6879(86)29101-6. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Higerd T. B., Spizizen J. Isolation of two acetyl esterases from extracts of Bacillus subtilis. J Bacteriol. 1973 Jun;114(3):1184–1192. doi: 10.1128/jb.114.3.1184-1192.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kawaguchi Y., Honda H., Taniguchi-Morimura J., Iwasaki S. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature. 1989 Sep 14;341(6238):164–166. doi: 10.1038/341164a0. [DOI] [PubMed] [Google Scholar]
  10. Koivula T. T., Hemilä H., Pakkanen R., Sibakov M., Palva I. Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius. J Gen Microbiol. 1993 Oct;139(10):2399–2407. doi: 10.1099/00221287-139-10-2399. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Manco G., Di Gennaro S., De Rosa M., Rossi M. Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius. Eur J Biochem. 1994 May 1;221(3):965–972. doi: 10.1111/j.1432-1033.1994.tb18812.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  17. Matsunaga A., Koyama N., Noso Y. Purification and properties of esterase from Bacillus stearothermophilus. Arch Biochem Biophys. 1974 Feb;160(2):504–513. doi: 10.1016/0003-9861(74)90427-5. [DOI] [PubMed] [Google Scholar]
  18. Matthews B. W. Structural and genetic analysis of protein stability. Annu Rev Biochem. 1993;62:139–160. doi: 10.1146/annurev.bi.62.070193.001035. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Peist R., Koch A., Bolek P., Sewitz S., Kolbus T., Boos W. Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity. J Bacteriol. 1997 Dec;179(24):7679–7686. doi: 10.1128/jb.179.24.7679-7686.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Reddy T. V., Weisburger E. K., Thorgeirsson S. S. Mutagenic activation of N-2-fluorenylacetamide and N-hydroxy-N-2-fluorenylacetamide in subcellular fractions from X/Gf mice. J Natl Cancer Inst. 1980 Jun;64(6):1563–1569. doi: 10.1093/jnci/64.6.1563. [DOI] [PubMed] [Google Scholar]
  24. Schmidt-Dannert C., Rúa M. L., Atomi H., Schmid R. D. Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. molecular cloning, nucleotide sequence, purification and some properties. Biochim Biophys Acta. 1996 May 31;1301(1-2):105–114. doi: 10.1016/0005-2760(96)00027-6. [DOI] [PubMed] [Google Scholar]
  25. Sobek H., Görisch H. Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem J. 1988 Mar 1;250(2):453–458. doi: 10.1042/bj2500453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  27. Varki A., Muchmore E., Diaz S. A sialic acid-specific O-acetylesterase in human erythrocytes: possible identity with esterase D, the genetic marker of retinoblastomas and Wilson disease. Proc Natl Acad Sci U S A. 1986 Feb;83(4):882–886. doi: 10.1073/pnas.83.4.882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vihinen M. Relationship of protein flexibility to thermostability. Protein Eng. 1987 Dec;1(6):477–480. doi: 10.1093/protein/1.6.477. [DOI] [PubMed] [Google Scholar]
  29. Watanabe K., Chishiro K., Kitamura K., Suzuki Y. Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006. J Biol Chem. 1991 Dec 25;266(36):24287–24294. [PubMed] [Google Scholar]
  30. 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]
  31. Wood A. N., Fernandez-Lafuente R., Cowan D. A. Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity. Enzyme Microb Technol. 1995 Sep;17(9):816–825. doi: 10.1016/0141-0229(94)00116-9. [DOI] [PubMed] [Google Scholar]
  32. Wrba A., Schweiger A., Schultes V., Jaenicke R., Závodszky P. Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. Biochemistry. 1990 Aug 21;29(33):7584–7592. doi: 10.1021/bi00485a007. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Zale S. E., Klibanov A. M. Why does ribonuclease irreversibly inactivate at high temperatures? Biochemistry. 1986 Sep 23;25(19):5432–5444. doi: 10.1021/bi00367a014. [DOI] [PubMed] [Google Scholar]

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