RT Journal Article SR Electronic(1) A1 Pérez, Dolores A1 Kovačić, Filip A1 Wilhelm, Susanne A1 Jaeger, Karl-Erich A1 García, María Teresa A1 Ventosa, Antonio A1 Mellado, EncarnaciónYR 2012 T1 Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus JF Microbiology, VO 158 IS 8 SP 2192 OP 2203 DO https://doi.org/10.1099/mic.0.058792-0 PB Microbiology Society, SN 1465-2080, AB 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 His6-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 (S72-M-T-K75) 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-S321-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.058792-0