Biosynthesis of 3,6-Dideoxy-heptoses for the Capsular Polysaccharides of Campylobacter jejuni

Abstract

Campylobacter jejuni is the leading cause of food poisoning in the United States. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. The CPS is composed of a repeating sequence of common and unusual sugar residues, including relatively rare heptoses. In the HS:5 serotype, we identified four enzymes required for the biosynthesis of GDP-3,6-dideoxy-β-l-ribo-heptose. In the first step, GDP-d-glycero-α-d-manno-heptose is dehydrated to form GDP-6-deoxy-4-keto-α-d-lyxo-heptose. This product is then dehydrated by a pyridoxal phosphate-dependent C3-dehydratase to form GDP-3,6-dideoxy-4-keto-α-d-threo-heptose before being epimerized at C5 to generate GDP-3,6-dideoxy-4-keto-β-l-erythro-heptose. In the final step, a C4-reductase uses NADPH to convert this product to GDP-3,6-dideoxy-β-l-ribo-heptose. These results are at variance with the previous report of 3,6-dideoxy-d-ribo-heptose in the CPS from serotype HS:5 of C. jejuni. We also demonstrated that GDP-3,6-dideoxy-β-l-xylo-heptose is formed using the corresponding enzymes found in the gene cluster from serotype HS:11 of C. jejuni. The utilization of different C4-reductases from other serotypes of C. jejuni enabled the formation of GDP-3,6-dideoxy-α-d-arabino-heptose and GDP-3,6-dideoxy-α-d-lyxo-heptose.

Figure 1

Structures of the repeating sugars in the CPSs from serotypes HS:15 and HS:19 of C. jejuni. The CPS from the HS:15 serotype contains l-arabinose and 6-deoxy-l-gulo-heptose, whereas the CPS from the HS:19 serotype contains the serinol amide of d-glucuronate and N-acetyl d-glucosamine. The N-acetyl d-glucosamine moiety from the CPS from HS:19 may also be modified nonstoichiometrically at C4 with a methyl phosphoramidate group.

Figure 2

Biosynthetic pathways for the formation of GDP-d-glycero-β-l-gluco-heptose (4) and GDP-6-deoxy-α-d-altro-heptose (7) and the initially proposed pathway for the biosynthesis of GDP-3,6-dideoxy-α-d-ribo-heptose (10).

Figure 3

Portion of the gene cluster from the HS:5 serotype of C. jejuni that is required for the biosynthesis of the 3,6-dideoxy-heptose moiety of the CPS. The individual genes are not drawn to the appropriate relative length.

Figure 4

SSN for the C3-dehydratases from C. jejuni. The closest 1000 sequences to the C3-dehydratase from C. jejuni serotype HS:5 at a sequence identity cutoff of 65%. The sequences for the 3-dehydratases from E. coli O55:H7 and Y. pseudotuberculosis IVA are shown in pink. The green and blue circles represent the apparent C3-dehydratases from various Campylobacter species and C. jejuni, respectively. The yellow circles represent the C3-dehydratases from serotypes HS:5, HS:11, and HS:45.

Figure 5

¹H NMR spectra of GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8) produced with the C3-dehydratase from serotype HS:5. (A) Reaction conducted in H₂O. (B) Reaction conducted in D₂O. Resonances for the hydrogens labeled with an “R” correspond to the ribose moiety of GDP, while those labeled with an “H” correspond to those of the heptose moiety. The multiplet a ∼3.6 ppm is likely due to contamination of glycerol. α-KG is the other reaction product formed from l-glutamate. Additional details are provided in the text.

Figure 6

Proposed reaction mechanism for the PLP-dependent C3-dehydratase. The PLP is recycled back to PMP in the first half reaction with l-glutamate.

Figure 7

(A) Mass spectrometry analysis of the reaction catalyzed by the C3-dehydratase. GDP-d-glycero-α-d-manno-heptose (1) prior to the addition of enzyme. (B) Reaction product, GDP-6-deoxy-4-keto-α-d-lyxo-heptose (5), after the addition of the C4,6-dehydratase to compound 1. (C) Reaction product, GDP-3,6-dideoxy-4-keto-d-threo-heptose (8), after the addition of the C3-dehydratase from serotype HS:5 to compound 5. Reaction products 5 and 8 appear at an m/z of 616.08 and 600.08, respectively. (D) Reaction product, GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8), after the addition of the C3-dehydratase from serotype HS:5 to compound 5 in 50% [¹⁸O]-H₂O. (E) Reaction product, GDP-3,6-dideoxy-β-l-ribo-heptose (12), produced from compound 1 by the enzymatic activities of the C4,6-dehydratase, C3-dehydratase, and C4-reductase in 50% [¹⁸O]-H₂O.

Figure 8

SSN for 18 epimerases identified from 33 serotyped strains of C. jejuni at a sequence identity of 89%. The specific serotype is labeled in each circle. The nodes in green and blue colors represent the C3- and C3/C5-epimerases, respectively, that were previously tested and functionally annotated for catalytic activity, whereas the yellow color designates the epimerases from serotypes HS:5, HS:11, and HS:45 of unknown function.

Figure 9

Portion of the ¹H NMR spectra showing the anomeric hydrogen at C1 of products formed from GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8) after the addition of an epimerase from either serotype HS:5 or HS:2. (A) GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8), (B) GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8) after the addition of epimerase from serotype HS:5, and (C) GDP-3,6-dideoxy-4-keto-α-d-threo-heptose (8) after the addition of an epimerase from serotype HS:2. The new triplet at 4.94 ppm is from the anomeric hydrogen at C1 of the product GDP-3,6-dideoxy-4-keto-β-l-erythro-heptose (11).

Figure 10

New biosynthetic pathways for the formation of GDP-3,6-dideoxy-β-l-ribo-heptose (12) and GDP-3,6-dideoxy-β-l-xylo-heptose (13).

Figure 11

¹H NMR spectra of GDP-3,6-dideoxy-β-l-ribo-heptose (12) using the C4-reductase from serotype HS:5. (A) Reaction conducted in H₂O. (B) Reaction conducted in D₂O. The loss of the resonances for C3 when the reaction was conducted in D₂O likely reflects the combined activities of the C3-dehydratase and the C5-epimerase used in the preparation of compound 12. Resonances for the hydrogens labeled with an “R” correspond to the ribose moiety of GDP, while those labeled with an “H” correspond to those of the heptose moiety. Additional details are provided in the text.