Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni

Abstract

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3–C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD­(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

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.…”

Biosynthesis of d-glycero-l-gluco-Heptose in the Capsular Polysaccharides of Campylobacter jejuni

“…Previously, we showed that Cj1427 was functionally able to catalyze the conversion of GDP- d - glycero -α- d - manno -heptose ( 1 ) to GDP- d - glycero -4-keto-α- d - lyxo -heptose ( 2 ) . We showed that the isolated Cj1427 contained a tightly bound NADH cofactor with an occupancy of 85%. , Additionally, we showed that Cj1427 does not exchange the bound cofactor during catalytic turnover, but required an α-keto acid cosubstrate, preferably α-ketoglutarate, to reoxidize the bound NADH to NAD + such that Cj1427 could catalyze the oxidation of GDP- d - glycero -α- d - manno -heptose ( 1 ) to GDP- d - glycero -4-keto-α- d - lyxo -heptose ( 2 ).…”

“…Cofactors bound to Cj1428 were determined by FPLC anion exchange chromatography. The FPLC methodology used was outlined previously . Briefly, concentrated Cj1428 (1.0 mM) was diluted with water to a final concentration of 400 μM and then heat-denatured (95 °C for 60 s) to liberate the bound cofactor.…”

“…The proposed transformation from this heptose to GDP- d - glycero -β- l - gluco -heptose ( 4 ) is illustrated in Scheme . , This biosynthetic pathway was based, in part, on demonstrated catalytic activities of Cj1430 and Cj1428 with surrogate substrates that lacked the hydroxyl group of the physiological substrate at C6 . However, we recently demonstrated that Cj1427 can effectively oxidize C4 of GDP- d - glycero -α- d - manno -heptose ( 1 ) in the presence of α-ketoglutarate (α-KG) to form GDP- d - glycero -4-keto-α- d - lyxo -heptose ( 2 ). , …”

Structure–function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways

“…In the Ddah pathway, the initiating C4, C6 dehydratase DdahA creates the 4-keto function and is specific for the C7 sugar ( 2 , 4 ). The initiating C4 oxidase for the Mlgh pathway MlghA was recently identified as Cj1427 (formerly WcaG), which requires α-ketoglutarate to support its activity on GDP- manno -heptose ( 29 , 30 ).…”