Academic Journal
Expanding the enzyme repertoire for sugar nucleotide epimerization : the CDP-tyvelose 2-epimerase from Thermodesulfatator atlanticus for glucose/mannose interconversion
| العنوان: | Expanding the enzyme repertoire for sugar nucleotide epimerization : the CDP-tyvelose 2-epimerase from Thermodesulfatator atlanticus for glucose/mannose interconversion |
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| المؤلفون: | Rapp, Christian, Van Overtveldt, Stevie, Beerens, Koen, Weber, Hansjörg, Desmet, Tom, Nidetzky, Bernd |
| المصدر: | APPLIED AND ENVIRONMENTAL MICROBIOLOGY ; ISSN: 0099-2240 ; ISSN: 1098-5336 |
| سنة النشر: | 2021 |
| المجموعة: | Ghent University Academic Bibliography |
| مصطلحات موضوعية: | Earth and Environmental Sciences, Biology and Life Sciences, Biotechnology, Food Science, Ecology, Applied Microbiology and Biotechnology, CDP-glucose, carbohydrate synthesis, catalytic mechanism, epimerase, multistep enzyme catalysis, short-chain dehydrogenase/reductase (SDR), specificity, sugar nucleotide, CYTIDINE DIPHOSPHATE 3, 6-DIDEOXYHEXOSES, UDP-GALACTOSE 4-EPIMERASE, SUBSTRATE-SPECIFICITY, CRYSTAL-STRUCTURE, YERSINIA-PSEUDOTUBERCULOSIS, CARBOHYDRATE EPIMERASES, STRUCTURAL-ANALYSIS, BIOSYNTHESIS, PURIFICATION, REDUCTASE |
| الوصف: | Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C-2 epimerization in all nucleotide (CDP, UDP, GDP, ADP, and TDP)-activated forms of D-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD(+)/subunit and shows optimum activity at 70 degrees C and pH 9.5. The enzyme exhibits a k(cat) with CDP-D-glucose of -1.0 min(-1) (pH 7.5 and 60 degrees C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemoenzymatic synthesis for CDP-D-mannose, CDP-D-deoxy-D-glucose, CDP-3-deoxy-o-glucose, and CDP-6-deoxy-D-xylo-hexopyranos-4-ulose. Attempts to obtain CDP-D-paratose and CDP-D-tyvelose were not successful. Using high-resolution carbohydrate analytics and in situ nuclear magnetic resonance (NMR) to monitor the enzymatic conversions (60 degrees C and pH 7.5), we show that the CDP-D-mannose/CDP-D-glucose ratio at equilibrium is 0.67 (+/- 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C-6 enhances the enzyme activity 5-fold compared to CDP-D-glucose, whereas deoxygenation at C-3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDRs (short-chain dehydrogenases/reductases), for which the current study now provides functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis. IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest ... |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | application/pdf |
| اللغة: | English |
| Relation: | https://biblio.ugent.be/publication/8693374; http://hdl.handle.net/1854/LU-8693374; http://dx.doi.org/10.1128/aem.02131-20; https://biblio.ugent.be/publication/8693374/file/8693375 |
| DOI: | 10.1128/aem.02131-20 |
| الاتاحة: | https://biblio.ugent.be/publication/8693374 http://hdl.handle.net/1854/LU-8693374 https://doi.org/10.1128/aem.02131-20 https://biblio.ugent.be/publication/8693374/file/8693375 |
| Rights: | Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) ; info:eu-repo/semantics/openAccess |
| رقم الانضمام: | edsbas.E01FDC42 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1128/aem.02131-20 |
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