https://repository.kopri.re.kr/handle/201206/11905 Tue, 21 Apr 2026 10:57:23 GMT 2026-04-21T10:57:23Z https://repository.kopri.re.kr/handle/201206/13714 Title: Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae Authors: Do, Hackwon; Wang, Ying; Lee, Chang Woo; Yoo, Wanki; Jeon, Sangeun; Hwang, Jisub; Lee, Min Ju; Kim, Kyeong Kyu; Kim, Han-Woo; Lee, Jun Hyuck; Kim, T. Doohun 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 exhib-ited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism un-derlying 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 dif-fraction dataset was collected up to 2.0 A resolution. The crystal structure was successfully deter-mined 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. Sat, 01 Jan 2022 00:00:00 GMT https://repository.kopri.re.kr/handle/201206/13714 2022-01-01T00:00:00Z https://repository.kopri.re.kr/handle/201206/13326 Title: Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4 Authors: Kwon, Sunghark; Ha, Hyun Ji; Kang, Yong Jun; Sung, Ji Hye; Hwang, Jisub; Lee, Min Ju; Lee, Jun Hyuck; Park, Hyun Ho Abstract: Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 A resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site. Fri, 31 Dec 2021 00:00:00 GMT https://repository.kopri.re.kr/handle/201206/13326 2021-12-31T00:00:00Z