Manganese-Induced Substrate Promiscuity in the Reaction Catalyzed by Phosphoglutamine Cytidylyltransferase from Campylobacter jejuni

Abstract

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-Lglutamine. 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.

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

“…jejuni NCTC 11168 is composed of 35 genes, as illustrated in Figure S1 . Previous investigations have functionally characterized the genes necessary for the biosynthesis of the d -glycero- l -gluco-heptose moiety and modification (Cj1152, Cj1423, Cj1424, Cj1425, Cj1427, Cj1428, and Cj1430). − In addition, the enzymes Cj1415, Cj1416, Cj1417, and Cj1418 have been shown to be required for the biosynthesis of the phosphoramidate modification, and gene knockout experiments have identified the enzymes (Cj1422 and Cj1421) required for the transfer of the phosphoramidate modifications to specific sugar receptors. ,− More recently, we have shown that Cj1441 is required for the conversion of uridine diphosphate (UDP)-glucose to UDP-glucuronate and that this enzyme is unable to catalyze the formation of an amide bond via the attack of an amine substrate with the putative thioester intermediate formed during the oxidation of UDP-glucose . The enzymes required for the biosynthesis of the two amines (serinol and ethanolamine) found in the HS:2 capsule are currently unknown.…”

Revised Model for the Type A Glycan Biosynthetic Pathway in Clostridioides difficile Strain 630Δerm Based on Quantitative Proteomics of cd0241–cd0244 Mutant Strains

“…To the best of our knowledge, CDP- l -threonine would be a so far not described cellular metabolite. However, several studies have shown the existence of amino acid residues linked to CDP in other prokaryotes, namely CDP- l -glutamine and CDP- l -serine . Furthermore, we predict CD0244 to be a CDP-threonine:​GlcNAc threonine phospho­transferase.…”

Functional and Structural Characterization of the UDP-Glucose Dehydrogenase Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni

“…These proteins include the eight enzymes known to be responsible for the Omethyl phosphoramidate decoration of the capsule [Cj1415− Cj1421 in C. jejuni NCTC 11168 (HS:2) and HS19.01− HS19.07 in C. jejuni NCTC 12517 (HS: 19)]. [9][10][11][12][13]22,49 Except for HS19.07 and Cj1421/Cj1422, the sequence identities for these proteins are ≥97%. The other relevant homologous enzymes include Cj1435 (HAD phosphatase), Cj1437 (PLPdependent aminotransferase), and Cj1441 (UDP-glucose 6dehydrogenase) where the amino acid sequence identity is >55%.…”

“…Comparison of the gene clusters for the biosynthesis of the capsular polysaccharides in C. jejuni NCTC 11168 (HS:2) and C. jejuni NCTC 12517 (HS:19) highlights the production of enzymes of nearly identical function (Table ). These proteins include the eight enzymes known to be responsible for the O -methyl phosphoramidate decoration of the capsule [Cj1415–Cj1421 in C. jejuni NCTC 11168 (HS:2) and HS19.01–HS19.07 in C. jejuni NCTC 12517 (HS:19)]. − ,, Except for HS19.07 and Cj1421/Cj1422, the sequence identities for these proteins are ≥97%. The other relevant homologous enzymes include Cj1435 (HAD phosphatase), Cj1437 (PLP-dependent aminotransferase), and Cj1441 (UDP-glucose 6-dehydrogenase) where the amino acid sequence identity is >55%.…”

“… , These sugars are further decorated by methylation, phosphoramidylation, and amidylation. The catalytic activities of four enzymes (Cj1415–Cj1418) that are partially responsible for the phosphoramidate modification in C. jejuni NCTC 11168 have been elucidated. − We, and others, have functionally characterized seven other enzymes that are required for the biosynthesis of the d - glycero - l - gluco -heptose moiety (Cj1152, Cj1423–Cj1425, Cj1427, Cj1428, and Cj1430) in C. jejuni NCTC 11168. − Knockout studies have shown that when the gene for Cj1423 from C. jejuni NCTC 11168 is deleted, the heptose sugar is no longer present in the capsular polysaccharide. ,, In the same study, a knockout of the gene for Cj1441 resulted in a complete loss of the capsule. The gene cluster that encodes most, but not all, of the enzymes required for the biosynthesis of the CPS of C. jejuni NCTC 11168 is presented in Figure .…”