Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni

Abstract

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Reaction Mechanism and Three-Dimensional Structure of GDP-d-glycero-α-d-manno-heptose 4,6-Dehydratase from Campylobacter jejuni

“…jejuni infections leads to the development of Guillain-Barré syndrome, an acquired autoimmune disorder resulting in muscle weakness, paralysis, and hospitalization . It is estimated that the various strains of Campylobacter cause over 25,000 deaths annually in children under five years of age, primarily affecting Asian and African populations . An important virulence factor for C.…”

“…At least 12 CPS structures from various strains of C. jejuni have been reported, and nine of them contain heptose moieties with at least 13 different structural variations. − Bioinformatic analyses and other experimental data suggest that all of the known heptose variations in C. jejuni likely originate from GDP- d -glycero-α- d -manno-heptose ( 3 ) .…”

“…7 It is estimated that the various strains of Campylobacter cause over 25,000 deaths annually in children under five years of age, primarily affecting Asian and African populations. 8 An important virulence factor for C. jejuni infections is the capsular polysaccharide (CPS), which coats the exterior cell surface and mediates the interactions of the bacterium with its host. 9 The CPSs vary in sugar composition and linkage within the various strains of the bacterium.…”

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

“…14 ethanolamine phosphate (8), whereas Cj1437 is a PLPdependent transaminase that catalyzes the stereospecific transamination of dihydroxyacetone phosphate to (S)-serinol phosphate (7). 15 The gene clusters for the biosynthesis of the CPSs made by C. jejuni NCTC 11168 (HS:2) and C. jejuni RM1285 (HS:19) are shown in Figure S1. Initially, attempts were made to catalyze amide bond formation using Cj1441, the UDP-glucose 6-dehydrogenase.…”

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates

“…5B and C). The expected product of DHAP transamination is serinol phosphate [43][44][45], and indeed, a compound with the mass of serinol phosphate was observed by LC-MS to accumulate in the reaction mixture (Supplementary Fig. 6).…”