Discovery and Functional Characterization of a Clandestine ATP-Dependent Amidoligase in the Biosynthesis of the Capsular Polysaccharide from Campylobacter jejuni.

Abstract

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that is commensal in poultry. Infection of C. jejuni leads to campylobacteriosis, the leading cause of gastroenteritis worldwide. Coating the surface of C. jejuni is a thick layer of sugar molecules known as the capsular polysaccharide (CPS). The CPS of C. jejuni NCTC 11168 (HS:2) is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronate, d-N-acetyl-galactosamine, and d-ribose. The glucuronate is further amidated with either ethanolamine or serinol, but it is unknown how this new amide bond is formed. Sequence similarity networks were used to identify a candidate enzyme for amide bond formation during the biosynthesis of the CPS of C. jejuni. The C-terminal domain of Cj1438 was shown to catalyze amide bond formation using MgATP and d-glucuronate in the presence of either ethanolamine phosphate or (S)-serinol phosphate. Product formation was verified using 31P NMR spectroscopy and ESI mass spectrometry, and the kinetic constants determined using a coupled enzyme assay by measuring the rate of ADP formation. This work represents the first functional characterization of an ATP-dependent amidoligase in the formation of amide bonds in the biosynthetic pathway for the assembly of the CPS in C. jejuni.