Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

• Published in: Microbiology (Society for General Microbiology) Vol. 158; no. Pt 8; pp. 2192 - 2203
• Main Authors: PEREZ, Dolores, KOVACIC, Filip, WILHELM, Susanne, JAEGER, Karl-Erich, GARCIA, María Teresa, VENTOSA, Antonio, MELLADO, Encarnación
• Format: Journal Article
• Language: English
• Published: Reading Society for General Microbiology 01.08.2012
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Catalysis; Catalytic Domain; Fundamental and applied biological sciences. Psychology; Hydrolysis; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Marinobacter - chemistry; Marinobacter - enzymology; Marinobacter - genetics; Microbiology; Molecular Sequence Data; Triacylglycerol lipase; Hydrolases; Esterases; Identification; Enzyme; Carboxylic ester hydrolases
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.058792-0

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.