Bifunctional Epimerase/Reductase Enzymes Facilitate the Modulation of 6-Deoxy-Heptoses Found in the Capsular Polysaccharides of Campylobacter jejuni

Abstract

Campylobacter jejuni is a human pathogen and the leading cause of food poisoning in the United States and Europe. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that consists of a repeating sequence of common and unusual carbohydrate segments. At least 10 different heptose sugars have thus far been identified in the various strains of C. jejuni. The accepted biosynthetic pathway for the construction of the 6-deoxy-heptoses begins with the 4,6-dehydration of GDP-d-glycero-d-manno-heptose by a dehydratase, followed by an epimerase that racemizes C3 and/or C5 of the product GDP-6-deoxy-4-keto-d-lyxo-heptose. In the final step, a C4-reductase catalyzes the NADPH reduction of the resulting 4-keto product. However, in some strains and serotypes of C. jejuni, there are two separate C4-reductases with different product specificities in the gene cluster for CPS formation. Five pairs of these tandem C4-reductases were isolated, and the catalytic properties were ascertained. In four out of five cases, one of the two C4-reductases is able to catalyze the isomerization of C3 and C5 of GDP-6-deoxy-4-keto-d-lyxo-heptose, in addition to the catalysis of the reduction of C4, thus bypassing the requirement for a separate C3/C5-isomerase. In each case, the 3′-end of the gene for the first C4-reductase contains a poly-G tract of 8–10 guanine residues that may be used to control the expression and/or catalytic activity of either C4-reductase. The three-dimensional structure of the C4-reductase from serotype HS:15, which only does a reduction of C4, was determined to 1.45 Å resolution in the presence of NADPH and GDP.

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

“…These results show conclusively that the product formed from the addition of the C5-epimerase and the C4-reductase from HS:5 is GDP-3,6-dideoxy-β- l - ribo -heptose ( 12 ) and that the product formed from the addition of the C5-epimerase and C4-reductase from HS:11 is GDP-3,6-dideoxy-β- l - xylo -heptose ( 13 ). These assignments are based on the known C4-reductase products from HS:2 and HS:15, and it is now apparent that these two C4-reductases are functionally able to catalyze the reduction of 3-deoxy substrates. , …”

“…The C3-dehydratases, epimerases, and C4-reductases from the HS:5 and HS:11 serotypes were purified according to the procedures reported previously. − Escherichia coli BL21­(DE3) competent cells were transformed by the appropriate plasmids. Single colonies were inoculated in 50 mL of LB medium (20 g/L of yeast extract, 35 g/L of tryptone, 5 g/L of sodium chloride, pH 7.0) supplemented with 50 μg/mL of kanamycin and grown at 37 °C overnight with shaking.…”

“…These assignments are based on the known C4-reductase products from HS:2 and HS:15, and it is now apparent that these two C4-reductases are functionally able to catalyze the reduction of 3-deoxy substrates. 14,15 Preparation of Previously Uncharacterized GDP-3,6dideoxy-heptoses. From the experiments denoted above, it is apparent that the C4-reductases from serotypes HS:2 and HS:15 can catalyze the reduction of the C4-keto group in GDP-3,6-dideoxy-4-keto-β-L-erythro-heptose (11) to generate either 12 or 13.…”

“…In each case, a new product was formed, and the NMR spectrum for each product is distinct from that obtained for either compound 12 or 13 (Figures S14 and S15). Based on the known product outcomes for the C4-reductase from HS:53 and HS:3, the two new products are GDP-3,6-dideoxy-α-D-arabino-heptose (14) and GDP-3,6-dideoxy-α-D-lyxo-heptose (15), respectively (Figure 10).…”

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

“…26 This ambiguity was solved when it was determined that all members of the r2 group of C4-reductases can catalyze the formation of product 14 directly from substrate 6, and thus these C4reductases can also epimerize C3 and C5 prior to the reduction of C4. 26 None of the other C4-reductases tested can epimerize either C3 or C5. 26 Biosynthesis of GDP-3,6-Dideoxy-heptoses.…”