Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential
| Autor: | Elena Jiménez-Ortega, Egle Narmontaite, Beatriz González-Pérez, Francisco J. Plou, María Fernández-Lobato, Julia Sanz-Aparicio |
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| Přispěvatelé: | Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Fundación Ramón Areces, ALBA Synchrotron, Jiménez-Ortega, Elena, Plou, Francisco J, Fernández-Lobato, María, Sanz-Aparicio, J., UAM. Departamento de Biología Molecular |
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
O-glycosylation
Sucrose β-fructofuranosidase glycosyl hydrolase family 32 X-ray crystallography mutagenesis transfructosylation activity fructooligosaccharides Organic Chemistry Hydrolase General Medicine Fructose Glycosyl hydrolase family 32 Biología y Biomedicina / Biología Catalysis Computer Science Applications Inorganic Chemistry Raffinose Mutagenesis Fructooligosaccharides Physical and Theoretical Chemistry Molecular Biology Transfctosylation activity Spectroscopy |
| Zdroj: | International Journal of Molecular Sciences; Volume 23; Issue 23; Pages: 14981 |
| ISSN: | 2016-7660 |
| Popis: | 17 pags., 5 figs., 1 tab. -- This article belongs to the Special Issue Protein Structure and Function in Microorganisms Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness through grants BIO2016-76601-C3-3-R/-C3-2-R/-C3-1-R, PID2019-105838RB-C33/-C32/C31 and Fundación Ramón Areces [XIX Call of Research Grants in Life and Material Sciences]. We are grateful to the staff of the Synchrotron Radiation Sources at Alba (Barcelona, Spain) for providing access and for technical assistance at BL13-XALOC beamline and to the Fundación Ramón Areces for an institutional grant to the Centre of Molecular Biology Severo Ochoa |
| Databáze: | OpenAIRE |
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