Biosynthesis of CDP-α-<scp>d</scp>-fucofuranose and CDP-β-<scp>l</scp>-6-deoxy-altrofuranose for the Capsular Polysaccharides of <i>Campylobacter jejuni</i>

<i>Campylobacter jejuni</i> is a Gram-negative human pathogen and is the most common cause of gastroenteritis in the United States and Europe. <i>C. jejuni</i> expresses a capsular polysaccharide (CPS) that enables the evasion of the host immune response and adherence to host epithelial cells. The various subspecies (serotypes) of <i>C. jejuni</i> are distinguished by their unique CPS repeating units. The repeating trisaccharide in the HS:41 serotype was previously found to contain l-arabinofuranose, 6-deoxy-d-<i>altro</i>-heptofuranose, and a third sugar, which is either d-fucofuranose or 6-deoxy-l-altrofuranose. Genome neighborhood and sequence similarity networks were employed to identify five candidate genes for the biosynthesis of d-fucofuranose and 6-deoxy-l-altrofuranose. Here, it was demonstrated that the biosynthetic pathways for both sugars are initiated by the formation of CDP-d-glucose from CTP and d-glucose-1-phosphate as catalyzed by HS41.21. This product is dehydrated by an NAD⁺-dependent 4,6-dehydratase (HS41.20) that produces CDP-4-keto-6-deoxy-d-glucose. The third enzyme (HS41.19) was shown to catalyze the inversion of stereochemistry at C3 and C5 using CDP-4-keto-6-deoxy-d-glucose as the substrate. The fourth enzyme (HS41.18) is an NADPH-dependent C4-reductase that catalyzes the formation of CDP-d-fucopyranose from CDP-4-keto-6-deoxy-d-glucose in the absence of the 3,5-epimerase but catalyzes the formation of CDP-6-deoxy-l-altropyranose in the presence of the epimerase. The last enzyme (HS41.17) in the pathway was shown to catalyze the FADH₂-dependent interconversion of CDP-d-fucopyranose and CDP-6-deoxy-l-altropyranose to CDP-d-fucofuranose and CDP-6-deoxy-l-altrofuranose, respectively. The overall synthesis of the two possible products is governed by the catalytic activity of the epimerase, since the C4-reductase and pyranose-furanose mutase are not affected by the stereochemistry at C5.