title: The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity.
abstract: The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, alpha- and beta-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20-80). It showed pronounced activity towards p-nitrophenyl and alpha- and beta-naphthyl esters of C(12)-C(16). Higher activity was observed with alpha-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 degrees C) and thermal stability. CD analysis revealed predominance of alpha-helical structure (54% alpha-helix, 21% beta-sheet) and a T(m) value at 66 degrees C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.
authors.affiliation: Laboratory of Molecular Genetics, Division of Molecular Biology, Ruder Bosković Institute, Bijenicka 54, 10000 Zagreb, Croatia.
authors.forename: Ana
authors.initials: A
authors.lastname: Bielen
authors.forename: Helena
authors.initials: H
authors.lastname: Cetković
authors.forename: Paul F
authors.initials: PF
authors.lastname: Long
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
authors.forename: Dusica
authors.initials: D
authors.lastname: Vujaklija
mesh_terms: D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D010957:Plasmids; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D006867:Hydrolases; D004950:Esterases; D008049:Lipase