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.

Metabolic Pathways for the Biosynthesis of Heptoses Used in the Construction of Capsular Polysaccharides in the Human Pathogen Campylobacter jejuni

“…In serotypes HS:5 and HS:11, the C5-epimerase inverts the stereochemistry at C5 to form GDP-3,6-dideoxy-4-keto-β- l - xylo -heptose ( 17 ). We have previously shown that the C4-reductases from HS:3 and HS:53 can reduce the 4-keto group of 16 to form the previously unknown compounds, GDP-3,6-dideoxy-α- d - arabino -heptose ( 20 ) and GDP-3,6-dideoxy-α- d - lyxo -heptose ( 21 ), respectively . These results demonstrate that the C4-reductases that appear in gene clusters for the biosynthesis of GDP-6-deoxy-heptoses can also function with GDP-4-keto-3,6-dideoxy substrates to generate 3,6-dideoxy heptoses that have not previously been identified within the CPS structures of C. jejuni .…”

“…The C4-keto functional group in GDP-6-deoxy-4-keto-α- d - lyxo -heptose ( 5 ) renders the stereochemical configuration at C3 and C5 susceptible to racemization via an enolate intermediate that is formed by proton abstraction from either C3 and/or C5. ,− Within the 33 common serotypes of C. jejuni , there are three types of C3- and C5-epimerases that have been identified thus far. ,,, The sequence similarity network (SSN) for the epimerases found within the gene clusters for heptose biosynthesis in C. jejuni is presented in Figure at a sequence identity cutoff of 89%. , The C3-epimerases have been shown to catalyze the epimerization of 6 to form GDP-6-deoxy-4-keto-α- d - arabino -heptose ( 7 ), whereas the C3/C5-epimerases have been shown to catalyze the epimerization of both C3 and C5 to generate an equilibrium mixture with GDP-6-deoxy-4-keto-β- l - ribo -heptose ( 8 ) and GDP-6-deoxy-4-keto-β- l - xylo -heptose ( 9 ), in addition to 7 . ,− Ten serotypes (HS:3, HS:4, HS:8, HS:10, HS:12, HS:23/36, HS:29, HS:33, HS:41, and HS:52) contain a C3-epimerase (91–99% identical to one another), whereas five of the serotypes (HS:2, HS:15, HS:32/HS:58, HS:42, and HS:63) have the gene for the C3/C5-epimerase (81–98% identical to one another). Five serotypes (HS:18, HS:27, HS:40, HS:51/HS:53, and HS:60) do not contain either a C3 or C3/C5-epimerase and are thus highly likely to utilize either GDP- d - glycero - d - manno -heptose ( 5 ) or GDP-6-deoxy- d - manno -heptose ( 10 ) in their ultimate capsular polysaccharide.…”

“…The putative C3-dehydratase (HS5.12) was shown to catalyze the conversion of 6 to GDP-3,6-dideoxy-4-keto- d - threo -heptose ( 16 ). This reaction was shown to require pyridoxal phosphate (PLP) and l -glutamate with the formation of α-ketoglutarate and ammonia . In the next step, the epimerase (HS5.14) catalyzes the racemization of C5 to form GDP-3,6-dideoxy-β- l - erythro -heptose ( 17 ).…”

“…Purification of the Cj1427 C4-dehydrogenase (HS:2), C3-dehydratase (HS:5), C3-epimerases (HS:3 and HS:23/36), C3/C5-epimerases (HS:2, HS:15, and HS:42), C5-epimerase (HS:11), and the various C4-reductases (HS:2, HS:3, HS:11, HS:15, HS:23/36, HS:33, HS:42 and HS:51/53) was conducted using the procedures reported previously. − In a typical purification, ∼10 g of frozen cell paste was resuspended in 100 mL of buffer A (50 mM HEPES, pH 8.0, 250 mM KCl, 5.0 mM imidazole) supplemented with 0.1 mg/mL lysozyme, 0.05 mg/mL protease inhibitor cocktail powder, and 40 U/mL DNase I. The suspended cells were lysed by sonication (Branson 450 Sonifier), and the supernatant solution was collected after centrifugation at 10,000 g for 30 min.…”

“…This reaction was shown to require pyridoxal phosphate (PLP) and L-glutamate with the formation of α-ketoglutarate and ammonia. 27 In the next step, the epimerase (HS5.14) catalyzes the racemization of C5 to form GDP-3,6-dideoxy-β-Lerythro-heptose (17). In the final step, the C4-reductase (HS5.13) catalyzes the NADPH-dependent reduction of 17 to GDP-3,6-dideoxy-β-L-ribo-heptose (18).…”