Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae

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.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production

“…The circular zones were generated as a result of ethyl ferulate hydrolysis. Previously characterized enzymes were used to examine feruloyl esterase activity [ 44 ]. La Fae activity was estimated using ethyl ferulate (1 mM) as a substrate; 10 µL of 1 mg/mL La Fae was added to 300 µL of reaction mixture.…”

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae

“…Lg EstI prefers the short acyl chain of p -nitrophenyl esters [ 19 ]. When different p -nitrophenyl esters with the acyl carbon group ranging from C 2 to C 12 were tested, only p NA was effectively cleaved by Lg EstI.…”

“…When different p -nitrophenyl esters with the acyl carbon group ranging from C 2 to C 12 were tested, only p NA was effectively cleaved by Lg EstI. Moreover, Lg EstI exhibited decreased activity (less than 10%) for p NH and p NO, and no activity against other substrates [ 19 ]. The thermostability of Lg EstI was tested every 15 min for 1 h at temperatures ranging from 20–60°C, revealing that the activity of Lg EstI was not significantly altered until 40°C; however, the activity was lowered to 50% after 60 min at 50°C ( Fig 1A ).…”

“…Crystallographic analysis of Lg EstI was conducted, as previously described [ 19 ]. The sitting-drop vapor diffusion method was used to crystallize Lg EstI.…”

Crystallographic Studies of Enzymes (Volume II)

“…Doohun Kim at Sookmyung Women's University and Drs. Han-Woo Kim, Hackwon Do and Jun Hyuck Lee at the Korea Polar Research Institute published several papers on esterase on a continuum of their previous work [1][2][3]. They identified new esterases from various microbial sources and characterized their unique enzymatic properties.…”