In vitro characterization of N-terminal truncated EpsC from Bacillus subtilis 168, a UDP-N-acetylglucosamine 4,6-dehydratase
| Autor: | K. Krishnamurthy Rao, Chinmayi R. Kaundinya, Handanahal S. Savithri, Petety V. Balaji |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
BIOCHEMICAL-CHARACTERIZATION Operon NAD(+) dependent UDP-GlcNAc 4 6-dehydratase PROTEIN Bacillus subtilis CAMPYLOBACTER Biochemistry Substrate Specificity chemistry.chemical_compound FUNCTIONAL-CHARACTERIZATION Nucleotide Chromatography High Pressure Liquid chemistry.chemical_classification biology Nucleoside Diphosphate Sugars PSEUDAMINIC ACID Recombinant Proteins Transmembrane domain Acetyltransferase GLCNAC Sequence analysis Biophysics 03 medical and health sciences Biosynthesis Bacterial Proteins Protein Domains Surface plasmon resonance Escherichia coli BIOSYNTHESIS DIAMINO SUGAR Molecular Biology Hydro-Lyases Enzyme Assays STAPHYLOCOCCUS-AUREUS N N ' - diacetylbacillosamine biosynthetic 030102 biochemistry & molecular biology PSEUDOMONAS-AERUGINOSA UDP-keto sugars biology.organism_classification Peptide Fragments carbohydrates (lipids) pathway enzyme Kinetics 030104 developmental biology chemistry Dehydratase Mutation |
| Zdroj: | IndraStra Global. |
| ISSN: | 2381-3652 |
| Popis: | Bacillus subtilis 168 EpsC is annotated as “Probable polysaccharide biosynthesis protein” in the SwissProt database. epsC is part of the eps operon, thought to be involved in the biosynthesis of exopolymeric substances (EPS). The present study was undertaken to determine the molecular function of EpsC. Sequence analysis of EpsC suggested the presence of a transmembrane domain. Two N-terminal deletion mutants in which residues 1–89 (EpsC89) and 1–115 (EpsC115) are deleted were cloned and overexpressed. Enzyme activity and substrate preferences were investigated by reverse phase HPLC, surface plasmon resonance (SPR) spectroscopy and absorption spectroscopy. These data show that EpsC has UDP-GlcNAc 4,6-dehydratase activity in vitro. Purified recombinant proteins were found to utilise UDP-Glc and TDP-Glc also as substrates. In addition, EpsC115 could utilise UDP-Gal and UDP-GalNAc as substrates whereas EpsC89 could only bind these two sugar nucleotides. These results show that deletion of a longer N-terminal region broadens substrate specificity. These broadened specificity is perhaps an outcome of the deletion of the putative transmembrane domain and may not be present in vivo. EpsC, together with the aminotransferase EpsN (Kaundinya CR et al., Glycobiology, 2018) and acetyltransferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N′-diacetylbacillosamine. |
| Databáze: | OpenAIRE |
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