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 31 P 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.

Functional Characterization of a HAD Phosphatase Involved in Capsular Polysaccharide Biosynthesis in Campylobacter jejuni

“…For example, the rates of hydrolysis of compounds S - 11 and 12 are >2 orders of magnitude faster than the rates of hydrolysis of compounds 3 and S - 4 . This observation is fully consistent with the fact that the enzyme responsible for amide bond formation does not utilize ethanolamine or serinol as a substrate . The amide bond forming enzyme Cj1438 does not utilize UDP- d -glucuronate as a substrate but can utilize d -glucuronate ( 5 ), the methyl glycoside of d -glucuronate ( 6 ), and the disaccharide with 1- O -methyl- d -ribose ( 13 ) .…”

“…It is known from our previous investigation that Cj1438 will not accept UDP- d -glucuronate as a substrate for the formation of the phosphorylated d -glucuronamide . This suggests that the glycosyltransferase for polysaccharide formation likely functions before the amidation reaction.…”

“…We have previously demonstrated that the C-terminal domain of Cj1438 catalyzes the ATP-dependent amidation of d -glucuronate ( 5 ) and the O -methyl glycoside of d -glucuronate ( 6 ) with either S -serinol phosphate ( S - 4 ) or ethanolamine phosphate ( 3 ) but will not utilize UDP- d -glucuronate as a substrate . These results imply that the physiological substrate for the phosphatase, Cj1435, is the phosphorylated d -glucuronamide moiety after it has been added to the growing polysaccharide chain.…”

“…The biosynthesis of the d -glucuronamide modification present in the CPS of C. jejuni is not completely understood. Previously, we have demonstrated that the enzymes Cj1441, Cj1436, and Cj1437 and the C-terminal domain of Cj1438 from C. jejuni NCTC 11168 (HS:2) are necessary for formation of the d -glucuronamides of S -serinol phosphate and ethanolamine phosphate. − The ultimate enzyme in the overall biosynthetic pathway is apparently Cj1435, which has been previously annotated as a HAD phosphatase. Here we demonstrated that Cj1435 is responsible for the hydrolysis of the phosphate group from the d -glucuronamide of either ethanolamine phosphate or S -serinol phosphate.…”

“…Cj1436 is a PLP-dependent enzyme that catalyzes the decarboxylation of l -serine phosphate to ethanolamine phosphate, and Cj1437 is a PLP-dependent transaminase that catalyzes the conversion of dihydroxyacetone phosphate to S -serinol phosphate using l -glutamate as the amine donor . The C-terminal domain of Cj1438 is an ATP-dependent amidoligase . This enzyme was shown to use MgATP, d -glucuronate (or the methyl glycoside of d -glucuronate), and either ethanolamine phosphate or S -serinol phosphate to synthesize the phosphorylated glucuronamide product .…”