Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i>
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)...
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MDPI AG
2021-12-01
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| author | Hackwon Do Ying Wang Chang Woo Lee Wanki Yoo Sangeun Jeon Jisub Hwang Min Ju Lee Kyeong Kyu Kim Han-Woo Kim Jun Hyuck Lee T. Doohun Kim |
| author_facet | Hackwon Do Ying Wang Chang Woo Lee Wanki Yoo Sangeun Jeon Jisub Hwang Min Ju Lee Kyeong Kyu Kim Han-Woo Kim Jun Hyuck Lee T. Doohun Kim |
| author_sort | Hackwon Do |
| collection | DOAJ |
| description | 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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| institution | Directory Open Access Journal |
| issn | 2073-4352 |
| language | English |
| last_indexed | 2024-03-10T01:41:13Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Crystals |
| spelling | doaj.art-48307a9259944ac1a820461529fe53ae2023-11-23T13:24:18ZengMDPI AGCrystals2073-43522021-12-011214610.3390/cryst12010046Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i>Hackwon Do0Ying Wang1Chang Woo Lee2Wanki Yoo3Sangeun Jeon4Jisub Hwang5Min Ju Lee6Kyeong Kyu Kim7Han-Woo Kim8Jun Hyuck Lee9T. Doohun Kim10Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaDepartment of Chemistry, Graduate School of General Studies, Sookmyung Women’s University, Seoul 04310, KoreaResearch Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaDepartment of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 16419, KoreaDepartment of Chemistry, Graduate School of General Studies, Sookmyung Women’s University, Seoul 04310, KoreaResearch Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaResearch Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaDepartment of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 16419, KoreaResearch Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaResearch Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, KoreaDepartment of Chemistry, Graduate School of General Studies, Sookmyung Women’s University, Seoul 04310, KoreaA 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.https://www.mdpi.com/2073-4352/12/1/46esterase<i>Lactococcus garvieae</i>X-ray crystallography |
| spellingShingle | Hackwon Do Ying Wang Chang Woo Lee Wanki Yoo Sangeun Jeon Jisub Hwang Min Ju Lee Kyeong Kyu Kim Han-Woo Kim Jun Hyuck Lee T. Doohun Kim Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> Crystals esterase <i>Lactococcus garvieae</i> X-ray crystallography |
| title | Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> |
| title_full | Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> |
| title_fullStr | Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> |
| title_full_unstemmed | Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> |
| title_short | Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (<i>Lg</i>EstI) from <i>Lactococcus garvieae</i> |
| title_sort | sequence analysis and preliminary x ray crystallographic analysis of an acetylesterase i lg i esti from i lactococcus garvieae i |
| topic | esterase <i>Lactococcus garvieae</i> X-ray crystallography |
| url | https://www.mdpi.com/2073-4352/12/1/46 |
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