1887

Abstract

The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K β-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the β-lactamase motif (S-M-T-K) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.

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2012-08-01
2019-10-21
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References

  1. Altschul S. F. , Madden T. L. , Schäffer A. A. , Zhang J. , Zhang Z. , Miller W. , Lipman D. J. . ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. . Nucleic Acids Res 25:, 3389–3402. [PubMed] [CrossRef]
    [Google Scholar]
  2. Arpigny J. L. , Jaeger K.-E. . ( 1999; ). Bacterial lipolytic enzymes: classification and properties. . Biochem J 343:, 177–183. [CrossRef] [PubMed]
    [Google Scholar]
  3. Bebrone C. , Moali C. , Mahy F. , Rival S. , Docquier J. D. , Rossolini G. M. , Fastrez J. , Pratt R. F. , Frère J. M. , Galleni M. . ( 2001; ). CENTA as a chromogenic substrate for studying β-lactamases. . Antimicrob Agents Chemother 45:, 1868–1871. [CrossRef] [PubMed]
    [Google Scholar]
  4. Bordo D. , Argos P. . ( 1991; ). Suggestions for “safe” residue substitutions in site-directed mutagenesis. . J Mol Biol 217:, 721–729. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bornscheuer U. T. . ( 2002; ). Microbial carboxyl esterases: classification, properties and application in biocatalysis. . FEMS Microbiol Rev 26:, 73–81. [CrossRef] [PubMed]
    [Google Scholar]
  6. Breuer M. , Ditrich K. , Habicher T. , Hauer B. , Kesseler M. , Stürmer R. , Zelinski T. . ( 2004; ). Industrial methods for the production of optically active intermediates. . Angew Chem Int Ed Engl 43:, 788–824. [PubMed] [CrossRef]
    [Google Scholar]
  7. Camacho C. , Coulouris G. , Avagyan V. , Ma N. , Papadopoulos J. , Bealer K. , Madden T. L. . ( 2009; ). blast+: architecture and applications. . BMC Bioinformatics 10:, 421. [CrossRef] [PubMed]
    [Google Scholar]
  8. Chahinian H. , Ali Y. B. , Abousalham A. , Petry S. , Mandrich L. , Manco G. , Canaan S. , Sarda L. . ( 2005; ). Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases. . Biochim Biophys Acta 1738:, 29–36.[PubMed] [CrossRef]
    [Google Scholar]
  9. DeLano W. L. . ( 2002; ). The PyMOL Molecular Graphics System. New York:: Schrodinger;.
    [Google Scholar]
  10. Elend C. , Schmeisser C. , Leggewie C. , Babiak P. , Carballeira J. D. , Steele H. L. , Reymond J. L. , Jaeger K.-E. , Streit W. R. . ( 2006; ). Isolation and biochemical characterization of two novel metagenome-derived esterases. . Appl Environ Microbiol 72:, 3637–3645. [PubMed] [CrossRef]
    [Google Scholar]
  11. Ewalt K. L. , Hendrick J. P. , Houry W. A. , Hartl F. U. . ( 1997; ). In vivo observation of polypeptide flux through the bacterial chaperonin system. . Cell 90:, 491–500. [CrossRef] [PubMed]
    [Google Scholar]
  12. Hall T. . ( 2007; ). BioEdit: Biological sequence alignment editor for Win95/98/NT/2K/XP/7, http://www.mbio.ncsu.edu/bioedit/bioedit.html.
  13. Hartl F. U. , Hayer-Hartl M. . ( 2002; ). Molecular chaperones in the cytosol: from nascent chain to folded protein. . Science 295:, 1852–1858. [CrossRef] [PubMed]
    [Google Scholar]
  14. Hasan F. , Shah A. A. , Hameed A. . ( 2006; ). Industrial applications of microbial lipases. . Enzyme Microb Technol 39:, 235–251. [CrossRef]
    [Google Scholar]
  15. Hausmann S. , Jaeger K.-E. . ( 2010; ). Lipolytic enzymes from bacteria. . In Handbook of Hydrocarbon and Lipid Microbiology, pp. 1099–1126. Edited by Timmis K. N. . . Berlin:: Springer;.[CrossRef]
    [Google Scholar]
  16. Holm C. , Davis R. C. , Osterlund T. , Schotz M. C. , Fredrikson G. . ( 1994; ). Identification of the active site serine of hormone-sensitive lipase by site-directed mutagenesis. . FEBS Lett 344:, 234–238. [PubMed] [CrossRef]
    [Google Scholar]
  17. Houde A. , Kademi A. , Leblanc D. . ( 2004; ). Lipases and their industrial applications: an overview. . Appl Biochem Biotechnol 118:, 155–170. [PubMed] [CrossRef]
    [Google Scholar]
  18. Huang Y. T. , Liaw Y. C. , Gorbatyuk V. Y. , Huang T. H. . ( 2001; ). Backbone dynamics of Escherichia coli thioesterase/protease I: evidence of a flexible active-site environment for a serine protease. . J Mol Biol 307:, 1075–1090. [CrossRef] [PubMed]
    [Google Scholar]
  19. Jaeger K.-E. , Eggert T. . ( 2002; ). Lipases for biotechnology. . Curr Opin Biotechnol 13:, 390–397. [CrossRef] [PubMed]
    [Google Scholar]
  20. Jaeger K.-E. , Holliger P. . ( 2010; ). Chemical biotechnology – a marriage of convenience and necessity. . Curr Opin Biotechnol 21:, 711–712. [CrossRef] [PubMed]
    [Google Scholar]
  21. Jaeger K.-E. , Reetz M. T. . ( 1998; ). Microbial lipases form versatile tools for biotechnology. . Trends Biotechnol 16:, 396–403. [CrossRef] [PubMed]
    [Google Scholar]
  22. Jeanmougin F. , Thompson J. D. , Gouy M. , Higgins D. G. , Gibson T. J. . ( 1998; ). Multiple sequence alignment with clustal x . . Trends Biochem Sci 23:, 403–405. [CrossRef] [PubMed]
    [Google Scholar]
  23. Jeon J. H. , Kim S. J. , Lee H. S. , Cha S. S. , Lee J. H. , Yoon S. H. , Koo B. S. , Lee C. M. , Choi S. H. . & other authors ( 2011; ). Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics. . Appl Environ Microbiol 77:, 7830–7836. [CrossRef] [PubMed]
    [Google Scholar]
  24. Joris B. , Ghuysen J. M. , Dive G. , Renard A. , Dideberg O. , Charlier P. , Frère J. M. , Kelly J. A. , Boyington J. C. . & other authors ( 1988; ). The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. . Biochem J 250:, 313–324.[PubMed]
    [Google Scholar]
  25. Kelley L. A. , Sternberg M. J. . ( 2009; ). Protein structure prediction on the Web: a case study using the Phyre server. . Nat Protoc 4:, 363–371. [CrossRef] [PubMed]
    [Google Scholar]
  26. Kelly J. A. , Kuzin A. P. . ( 1995; ). The refined crystallographic structure of a DD-peptidase penicillin-target enzyme at 1.6 Å resolution. . J Mol Biol 254:, 223–236. [CrossRef] [PubMed]
    [Google Scholar]
  27. Kim Y. H. , Kwon E. J. , Kim S. K. , Jeong Y. S. , Kim J. , Yun H. D. , Kim H. . ( 2010; ). Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library. . Biochem Biophys Res Commun 393:, 45–49. [CrossRef] [PubMed]
    [Google Scholar]
  28. Knox J. R. , Moews P. C. , Frere J. M. . ( 1996; ). Molecular evolution of bacterial β-lactam resistance. . Chem Biol 3:, 937–947. [CrossRef] [PubMed]
    [Google Scholar]
  29. Kuzin A. P. , Liu H. , Kelly J. A. , Knox J. R. . ( 1995; ). Binding of cephalothin and cefotaxime to d-Ala-d-Ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum β-lactamases. . Biochemistry 34:, 9532–9540. [CrossRef] [PubMed]
    [Google Scholar]
  30. Laane C. , Boeren S. , Vos K. , Veeger C. . ( 1987; ). Rules for optimization of biocatalysis in organic solvents. . Biotechnol Bioeng 30:, 81–87. [CrossRef] [PubMed]
    [Google Scholar]
  31. Laemmli U. K. . ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. . Nature 227:, 680–685. [CrossRef] [PubMed]
    [Google Scholar]
  32. Leščić Ašler I. , Ivić N. , Kovačić F. , Schell S. , Knorr J. , Krauss U. , Wilhelm S. , Kojić-Prodić B. , Jaeger K.-E. . ( 2010; ). Probing enzyme promiscuity of SGNH hydrolases. . ChemBioChem 11:, 2158–2167. [CrossRef] [PubMed]
    [Google Scholar]
  33. Lobkovsky E. , Moews P. C. , Liu H. , Zhao H. , Frère J. M. , Knox J. R. . ( 1993; ). Evolution of an enzyme activity: crystallographic structure at 2-Å resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. . Proc Natl Acad Sci U S A 90:, 11257–11261. [CrossRef] [PubMed]
    [Google Scholar]
  34. Martín S. , Márquez M. C. , Sánchez-Porro C. , Mellado E. , Arahal D. R. , Ventosa A. . ( 2003; ). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. . Int J Syst Evol Microbiol 53:, 1383–1387. [PubMed] [CrossRef]
    [Google Scholar]
  35. Mellado E. , Martín S. , Sánchez-Porro C. , Ventosa A. . ( 2005; ). Lipolytic enzymes from extremophilic microorganisms. . In Microorganisms for Industrial Enzymes and Biocontrol, pp. 25–43. Edited by Mellado E. , Barredo J. L. . . Kerala, India:: Research Signpost;.
    [Google Scholar]
  36. Mosbah H. , Sayari A. , Horchani H. , Gargouri Y. . ( 2007; ). Involvement of Gly 311 residue on substrate discrimination, pH and temperature dependency of recombinant Staphylococcus xylosus lipase: a study with emulsified substrate. . Protein Expr Purif 55:, 31–39. [CrossRef] [PubMed]
    [Google Scholar]
  37. Narayanan N. , Khan M. , Chou C. P. . ( 2011; ). Enhancing functional expression of heterologous Burkholderia lipase in Escherichia coli . . Mol Biotechnol 47:, 130–143. [CrossRef] [PubMed]
    [Google Scholar]
  38. Niehaus F. , Bertoldo C. , Kähler M. , Antranikian G. . ( 1999; ). Extremophiles as a source of novel enzymes for industrial application. . Appl Microbiol Biotechnol 51:, 711–729. [PubMed] [CrossRef]
    [Google Scholar]
  39. Nishihara K. , Kanemori M. , Kitagawa M. , Yanagi H. , Yura T. . ( 1998; ). Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli . . Appl Environ Microbiol 64:, 1694–1699.[PubMed]
    [Google Scholar]
  40. Nishihara K. , Kanemori M. , Yanagi H. , Yura T. . ( 2000; ). Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli . . Appl Environ Microbiol 66:, 884–889. [CrossRef] [PubMed]
    [Google Scholar]
  41. Oefner C. , D’Arcy A. , Daly J. J. , Gubernator K. , Charnas R. L. , Heinze I. , Hubschwerlen C. , Winkler F. K. . ( 1990; ). Refined crystal structure of β-lactamase from Citrobacter freundii indicates a mechanism for β-lactam hydrolysis. . Nature 343:, 284–288. [PubMed] [CrossRef]
    [Google Scholar]
  42. Ogino H. , Ishikawa H. . ( 2001; ). Enzymes which are stable in the presence of organic solvents. . J Biosci Bioeng 91:, 109–116.[PubMed] [CrossRef]
    [Google Scholar]
  43. Ollis D. L. , Cheah E. , Cygler M. , Dijkstra B. W. , Frolow F. , Franken S. M. , Harel M. , Remington S. J. , Silman I. . & other authors ( 1992; ). The α/β hydrolase fold. . Protein Eng 5:, 197–211. [CrossRef] [PubMed]
    [Google Scholar]
  44. Park J. H. , Ha H. J. , Lee W. K. , Généreux-Vincent T. , Kazlauskas R. J. . ( 2009; ). Molecular basis for the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates catalyzed by Candida antarctica lipase B. . ChemBioChem 10:, 2213–2222. [PubMed] [CrossRef]
    [Google Scholar]
  45. Pérez D. , Ventosa A. , Mellado E. , Guisán J. M. , Fernández-Lorente G. , Filice M. . ( 2010; ). Lipasa LipBL y sus aplicaciones. Spanish patent P201031636.
  46. Pérez D. , Martín S. , Fernández-Lorente G. , Filice M. , Guisán J. M. , Ventosa A. , García M. T. , Mellado E. . ( 2011; ). A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA). . PLoS ONE 6:, e23325.[PubMed] [CrossRef]
    [Google Scholar]
  47. Peters G. H. , Svendsen A. , Langberg H. , Vind J. , Patkar S. A. , Toxvaerd S. , Kinnunen P. K. . ( 1998; ). Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase. . Biochemistry 37:, 12375–12383. [PubMed] [CrossRef]
    [Google Scholar]
  48. Petersen E. I. , Valinger G. , Sölkner B. , Stubenrauch G. , Schwab H. . ( 2001; ). A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to β-lactamases and DD-peptidases. . J Biotechnol 89:, 11–25. [PubMed] [CrossRef]
    [Google Scholar]
  49. Rashamuse K. , Magomani V. , Ronneburg T. , Brady D. . ( 2009; ). A novel family VIII carboxylesterase derived from a leachate metagenome library exhibits promiscuous β-lactamase activity on nitrocefin. . Appl Microbiol Biotechnol 83:, 491–500. [CrossRef] [PubMed]
    [Google Scholar]
  50. Rodriguez J. A. , Mendoza L. D. , Pezzotti F. , Vanthuyne N. , Leclaire J. , Verger R. , Buono G. , Carriere F. , Fotiadu F. . ( 2008; ). Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases. . Anal Biochem 375:, 196–208. [CrossRef] [PubMed]
    [Google Scholar]
  51. Sambrook J. , Russell D. W. . ( 2001; ). Molecular Cloning: a Laboratory Manual, , 3rd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  52. Sambrook J. , Fritsch E. F. , Maniatis T. . ( 1989; ). Molecular Cloning: a Laboratory Manual, , 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  53. Sayari A. , Mosbah H. , Gargouri Y. . ( 2007; ). Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli . . Mol Biotechnol 36:, 14–22. [CrossRef] [PubMed]
    [Google Scholar]
  54. Schmid A. , Dordick J. S. , Hauer B. , Kiener A. , Wubbolts M. , Witholt B. . ( 2001; ). Industrial biocatalysis today and tomorrow. . Nature 409:, 258–268. [CrossRef] [PubMed]
    [Google Scholar]
  55. Shuo-Shuo C. , Xue-Zheng L. , Ji-Hong S. . ( 2011; ). Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948. . Protein Expr Purif 77:, 166–172. [CrossRef] [PubMed]
    [Google Scholar]
  56. Snellman E. A. , Colwell R. R. . ( 2004; ). Acinetobacter lipases: molecular biology, biochemical properties and biotechnological potential. . J Ind Microbiol Biotechnol 31:, 391–400. [CrossRef] [PubMed]
    [Google Scholar]
  57. Snellman E. A. , Sullivan E. R. , Colwell R. R. . ( 2002; ). Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1. . Eur J Biochem 269:, 5771–5779. [CrossRef] [PubMed]
    [Google Scholar]
  58. Thornton J. M. , Orengo C. A. , Todd A. E. , Pearl F. M. . ( 1999; ). Protein folds, functions and evolution. . J Mol Biol 293:, 333–342. [CrossRef] [PubMed]
    [Google Scholar]
  59. Tiquia S. M. , Mormile M. R. . ( 2010; ). Extremophiles – a source of innovation for industrial and environmental applications. . Environ Technol 31:, 823. [PubMed] [CrossRef]
    [Google Scholar]
  60. Valinger G. , Hermann M. , Wagner U. G. , Schwab H. . ( 2007; ). Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins. . J Biotechnol 129:, 98–108. [PubMed] [CrossRef]
    [Google Scholar]
  61. Wagner U. G. , Petersen E. I. , Schwab H. , Kratky C. . ( 2002; ). EstB from Burkholderia gladioli: a novel esterase with a β-lactamase fold reveals steric factors to discriminate between esterolytic and β-lactam cleaving activity. . Protein Sci 11:, 467–478. [PubMed] [CrossRef]
    [Google Scholar]
  62. Winkler U. K. , Stuckmann M. . ( 1979; ). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens . . J Bacteriol 138:, 663–670.[PubMed]
    [Google Scholar]
  63. Wu L. , Liu B. , Hong Y. , Sheng D. , Shen Y. , Ni J. . ( 2010; ). Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase. . Biotechnol Lett 32:, 107–112. [CrossRef] [PubMed]
    [Google Scholar]
  64. Yu E. Y. , Kwon M. A. , Lee M. , Oh J. Y. , Choi J. E. , Lee J. Y. , Song B. K. , Hahm D. H. , Song J. K. . ( 2011; ). Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome. . Appl Microbiol Biotechnol 90:, 573–581. [CrossRef] [PubMed]
    [Google Scholar]
  65. Zaccai G. . ( 2004; ). The effect of water on protein dynamics. . Philos Trans R Soc Lond B Biol Sci 359:, 1269–1275. [CrossRef] [PubMed]
    [Google Scholar]
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