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

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 (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 EpsC, together with the aminotransferase EpsN (Kaundinya CR et al., Glycobiology, 2018) and acetyltransferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N'-diacetylbacillosamine.

Keywords: N,N′ - diacetylbacillosamine biosynthetic pathway enzyme; NAD(+) dependent UDP-GlcNAc 4,6-dehydratase; Surface plasmon resonance; UDP-keto sugars.