| Summary: | A gene encoding <i>Lg</i>EstI was cloned from a bacterial fish pathogen, <i>Lactococcus garvieae</i>. Sequence and bioinformatic analysis revealed that <i>Lg</i>EstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from <i>Acanthamoeba polyphaga mimivirus</i> (APMV). Here, we present the results of <i>Lg</i>EstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type <i>Lg</i>EstI protein was overexpressed in <i>Escherichia coli</i>, and its enzymatic activity was tested using <i>p</i>-nitrophenyl of varying lengths. <i>Lg</i>EstI protein exhibited higher esterase activity toward <i>p</i>-nitrophenyl acetate. To better understand the mechanism underlying <i>Lg</i>EstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of <i>Lg</i>EstI. First, the wild-type <i>Lg</i>EstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (<i>w</i>/<i>v</i>) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of <i>Lg</i>EstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting <i>Lg</i>EstI to protein engineering.
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