Structure Report for: 7YC4

Do, H.,Lee, J.H.

Title: Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae
Do H, Yoo W, Wang Y, Nam Y, Shin SC, Kim HW, Kim KK, Lee JH
Ref: PLoS ONE, 18:e0280988, 2023 : PubMed
Abstract


ESTHER: Do_2023_PLoS.One_18_e0280988
PubMedSearch: Do 2023 PLoS.One 18 e0280988
PubMedID: 36745644
Gene_locus related to this paper: 9lact-a0a5m9r5n4

Abstract

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.



        

Title: Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae
Do H, Wang Y, Lee CW, Yoo W, Jeon S, Hwang J, Lee MJ, Kim KK, Kim HW and Kim TD <1 more author(s)> Do H, Wang Y, Lee CW, Yoo W, Jeon S, Hwang J, Lee MJ, Kim KK, Kim HW, Lee JH, Kim TD (- 1)
Ref: Crystals, 12:46, 2022 : PubMed
Abstract


ESTHER: Do_2022_Crystals_12_46
PubMedSearch: Do 2022 Crystals 12 46
Gene_locus related to this paper: 9lact-a0a5m9r5n4

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 A 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.