Biochemical and structural characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family
| Autor: | Jelena Rajkovic, Juan A. Hermoso, José Berenguer, Elisa Beneventi, Mercedes Sánchez-Costa, Aurelio Hidalgo, Jacobo Cruces, María-Isabel González-Siso, Ana Torrado, Ivanna Rivera, Vega Miguel-Ruano, María L. Rúa, Kamila Knapik, Manuel Becerra, José M. Otero |
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| Přispěvatelé: | ALBA Synchrotron, European Commission, Ministerio de Ciencia, Innovación y Universidades (España), UAM. Departamento de Biología Molecular |
| Rok vydání: | 2021 |
| Předmět: |
DMSO
dimethyl sulfoxide Biochemistry Esterase 0302 clinical medicine Structural Biology Enzime-Substrate Complex CHCA cyclohexane carboxylic acid CV column volume chemistry.chemical_classification 0303 health sciences biology Chemistry NP naproxen Biología y Biomedicina / Biología 2306 Química Orgánica pNP 4-nitrophenol 3. Good health Computer Science Applications DSF Differential scanning fluorimetry 030220 oncology & carcinogenesis Crystal structures Enzyme-substrate complex Crystal Structure 2301 Química Analítica HSL hormone-sensitive lipase Research Article Biotechnology CMC critical micellar concentration Hydrolase Fold Stereochemistry Biophysics FLU fluorescein 03 medical and health sciences Metagenomic PPL Porcine Pancreatic Lipase Genetics Lipase ComputingMethodologies_COMPUTERGRAPHICS 030304 developmental biology Enzyme substrate complex 2403 Bioquímica Thermophilic esterase Thermophile Substrate (chemistry) Active site LDAO N N-dimethyldodecylamine N-oxide Enzyme biology.protein α/β hydrolase fold MNP methyl-naproxen Linker TP248.13-248.65 |
| Zdroj: | Investigo. Repositorio Institucional de la Universidade de Vigo Universidade de Vigo (UVigo) Digital.CSIC. Repositorio Institucional del CSIC instname Biblos-e Archivo. Repositorio Institucional de la UAM RUC. Repositorio da Universidade da Coruña Computational and Structural Biotechnology Journal, Vol 19, Iss, Pp 1214-1232 (2021) Computational and Structural Biotechnology Journal RUC: Repositorio da Universidade da Coruña Universidade da Coruña (UDC) |
| Popis: | Graphical abstract Highlights • A novel thermophilic esterase discovered from hot-spring metagenomic library. • EstD11 shows broad substrate specificity including substrates of industrial interest. • Atomic resolution crystallographic complexes reveal hot-spots in the active site. • High-T structures provide unique evidence on cap dynamics as occurs in vivo. • Met zipper at active site essential in thermo-stability and substrate promiscuity. A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/β-hydrolase superfamily. The canonical α/β-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11. |
| Databáze: | OpenAIRE |
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