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Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-β-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-β-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 A in the presence of bound GDP-d-glycero-β-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 A. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP:phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by L-glutamine phosphate to form CDP-L-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn(2+), Cj1416 catalyzes the formation of 12 different reaction products using L-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, L-serine phosphate or 3-phospho-D-glycerate as the nucleophile to displace pyrophosphate from CTP.

YcjR from Escherichia coli K-12 MG1655 catalyzes the manganese-dependent reversible epimerization of 3-keto-α-D-gulosides to the corresponding 3-keto-α-D-glucosides as a part of a proposed catabolic pathway for the transformation of D-gulosides to D-glucosides. The three-dimensional structure of the manganese-bound enzyme was determined by X-ray crystallography. The divalent manganese ion is coordinated to the enzyme by ligation to Glu-146, Asp-179, His-205, and Glu-240. When either of the two active site glutamate residues are mutated to glutamine the enzyme loses all catalytic activity for the epimerization of α-methyl-3-keto-D-glucoside at C4. However, the E240Q mutant is able to catalyze hydrogen/deuterium exchange of the proton at C4 of α-methyl-3-keto-D-glucoside in solvent D(2)O. The E146Q mutant does not catalyze this exchange reaction. These results indicate that YcjR catalyzes the isomerization of 3-keto-D-glucosides via proton abstraction at C4 by Glu-146 to form a cis-enediolate intermediate that is subsequently protonated on the opposite face by Glu-240 to generate the corresponding 3-keto-D-guloside. This conclusion is supported by docking of the cis-enediolate intermediate into the active site of YcjR based on the known binding orientation of D-fructose and D-psicose in the active site of D-psicose-3-epimerase. It is proposed that YcjR be named 3-keto-D-glucoside-4-epimerase.

Campylobacter jejuni, a leading cause of gastroenteritis world wide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (L-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of L-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of L-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-P(β)-P(γ)) and phosphorylated (Enz-X-P(β)) intermediates. In the absence of L-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[(18)O(4)]-ATP in support of the formation of the Enz-X-P(β)-P(γ) intermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between L-glutamine phosphate and [(15)N-amide]-L-glutamine in direct support of the Enz-X-P(β) intermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of D-glutamine, γ-L-glutamyl hydroxamate, γ-L-glutamyl hydrazide and β-L-aspartyl hydroxamate, in addition to L-glutamine.

Campylobacter jejuni , a leading cause of gastroenteritis, produces a capsular polysaccharide that is derivatized with a unique O-methyl phosphoramidate (MeOPN) modification. This modification contributes to serum resistance and invasion of epithelial cells. Previously, the first three biosynthetic steps for the formation of MeOPN have been elucidated. The first step is catalyzed by a novel glutamine kinase (Cj1418), which catalyzes the ATP-dependent phosphorylation of the amide nitrogen of L-glutamine. L-Glutamine phosphate is used by CTP:phosphoglutamine cytidylyltransferase (Cj1416) to displace pyrophosphate from CTP to generate CDP-L-glutamine, which is then hydrolyzed by γ-glutamyl-CDP-amidate hydrolase (Cj1417) to form cytidine diphosphoramidate (CDP-NH(2)). Here we show that Cj1415 catalyzes the ATP-dependent phosphorylation of CDP-NH(2) to form 3′-phospho-cytidine-5′-diphosphoramidate. Cj1415 will also catalyze the phosphorylation of adenosine diphosphoramidate (ADP-NH(2)) and uridine diphosphoamidate (UDP-NH(2)) but at significantly reduced rates. It is proposed that Cj1415 be named cytidine diphosphoramidate kinase.

Campylobacter jejuni is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. C. jejuni produces a capsular polysaccharide (CPS) that contains a unique O-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of L-glutamine to form L-glutamine phosphate. In this investigation the metabolic fate of L-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by L-glutamine phosphate to form CDP-L-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-L-glutamine to generate cytidine diphosphoramidate and L-glutamate. The structures of the two novel intermediates, CDP-L-glutamine and cytidine diphosphoramidate, were confirmed by 31P NMR spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP: phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase.