In vitro characterization of N-terminal truncated EpsC from Bacillus subtilis 168, a UDP-N-acetylglucosamine 4,6-dehydratase

Kaundinya, Chinmayi R and Savithri, Handanahal S and Rao, K Krishnamurthy and Balaji, Petety V (2018) In vitro characterization of N-terminal truncated EpsC from Bacillus subtilis 168, a UDP-N-acetylglucosamine 4,6-dehydratase. In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 657 . pp. 78-88.

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Abstract

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 (EpsC(89)) and 1-115 (EPsC(115)) 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, EpsC(115) could utilise UDP-Gal and UDP-GalNAc as substrates whereas EpsC(89) 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 EpsC, together with the aminotransferase EpsN (Kaundinya CR et aL , Glycobiology, 2018) and acetyl-transferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N'-diacetylbacillosamine.

Item Type:	Journal Article
Publication:	ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Publisher:	ELSEVIER SCIENCE INC
Additional Information:	Copy right for this article belong to ELSEVIER SCIENCE INC
Keywords:	UDP-keto sugars; NAD(+) dependent UDP-GlcNAc 4,6-dehydratase; N,N ` - diacetylbacillosamine biosynthetic; pathway enzyme; Surface plasmon resonance
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	13 Nov 2018 15:26
Last Modified:	13 Nov 2018 15:26
URI:	http://eprints.iisc.ac.in/id/eprint/61023

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