Structure–function insights into the fungal endo-chitinase Chit33 depict its mechanism on chitinous material
Entidad
UAM. Departamento de Biología MolecularEditor
MDPIFecha de edición
2022-07-02Cita
10.3390/ijms23147599
International Journal of Molecular Sciences 23.14 (2022): 7599
ISSN
1661-6596 (print); 1422-0067 (online)DOI
10.3390/ijms23147599Proyecto
Gobierno de España. PID2019-105838RB-C33; Gobierno de España. PID2019-105838RB-C32Versión del editor
https://doi.org/10.3390/ijms23147599Materias
Arginine; Aspartic Acid; Hydrolase; Tyrosine; Mutant Protein; Biología y Biomedicina / BiologíaDerechos
© 2022 by the authorsResumen
Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type endo-chitinase Chit33 from Trichoderma harzianum and its D165A/E167A-Chit33-(NAG)4 complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite –1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants’. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation
Lista de ficheros
Google Scholar:Jiménez-Ortega, Elena
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Kidibule, Peter Elias
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Fernández Lobato, María
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Sanz-Aparicio, Julia
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