title: Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus.
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 Marinobacter lipolyticus. 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(6)-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(72)-M-T-K(75)) 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(321)-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Pérez
authors.affiliation: Institute for Molecular Enzyme Technology, Heinrich-Heine University, Düsseldorf, Forschungszentrum Jülich, Germany.
authors.forename: Filip
authors.initials: F
authors.lastname: Kovačić
authors.affiliation: Institute for Molecular Enzyme Technology, Heinrich-Heine University, Düsseldorf, Forschungszentrum Jülich, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilhelm
authors.affiliation: Institute for Molecular Enzyme Technology, Heinrich-Heine University, Düsseldorf, Forschungszentrum Jülich, Germany.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: García
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ventosa
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain.
authors.forename: Encarnación
authors.initials: E
authors.lastname: Mellado
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053520:Marinobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D008049:Lipase