Abstract
A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) 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 LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.
Highlights
Esterases (E.C. 3.1.1.X) catalyze the hydrolysis of various substrates containing ester groups
Extensive efforts are being made to identify unique esterases with higher activity, improved stability, and broad substrate specificity from newly found microorganism genomes as well as metagenomes. Such esterases can be further subjected to protein engineering to generate esterases with a precise structure and desirable functions [3]
The LgEstI sequence was blasted against the Protein Data Bank (PDB) database, and the result (E-value cutoff for reporting = 1 × 10−10) was reloaded for performing a second blast against the Uniprot_sport database
Summary
Esterases (E.C. 3.1.1.X) catalyze the hydrolysis of various substrates containing ester groups. Esterases are serine hydrolases that contain a conserved Ser-Asp/Glu-His catalytic triad with an α/β hydrolase fold. Esterases harbor the same α/β hydrolase fold and have high sequence homology, they have different substrate specificities and perform varying biological functions. Extensive efforts are being made to identify unique esterases with higher activity, improved stability, and broad substrate specificity from newly found microorganism genomes as well as metagenomes. Such esterases can be further subjected to protein engineering to generate esterases with a precise structure and desirable functions [3]
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