C3- and C3/C5-Epimerases Required for the Biosynthesis of the Capsular Polysaccharides from Campylobacter jejuni

Abstract

Campylobacter jejuni is a human pathogen and one of the leading causes of food poisoning in Europe and the United States. The outside of the bacterium is coated with a capsular polysaccharide that assists in the evasion of the host immune system. Many of the serotyped strains of C. jejuni contain a 6-deoxy-heptose moiety that is biosynthesized from GDP-D-glycero-D-mannoheptose by the successive actions of a 4,6-dehydratase, a C3/C5-epimerase, and a C4-reductase. We identified 18 different C3/C5epimerases that could be clustered together into three groups at a sequence identity of >89%. Four of the enzymes from the largest cluster (from serotypes HS:3, HS:10, HS:23/36, and HS:41) were shown to only catalyze the epimerization at C3. Three enzymes from the second largest cluster (HS:2, HS:15, and HS:42) were shown to catalyze the epimerization at C3 and C5. Enzymes from the third cluster were not characterized. The three-dimensional structures of the epimerases from serotypes HS:3, HS:23/36, HS:15, and HS:41 were determined to resolutions of 1.5−1.9 Å. The overall subunit architecture places these enzymes into the diverse "cupin" superfamily. Within X-ray coordinate error, the immediate regions surrounding the active sites are identical, suggesting that factors extending farther out may influence product outcome. The X-ray crystal structures are consistent with His-67 and Tyr-134 acting as general acid/base catalysts for the epimerization of C3 and/or C5. Two amino acid changes (A76V/C136L) were enough to convert the C3-epimerase from serotype HS:3 to one that could now catalyze the epimerization at both C3 and C5.

Product Specificity of C4-Reductases in the Biosynthesis of GDP-6-Deoxy-Heptoses during Capsular Polysaccharide Formation in Campylobacter jejuni

“…The steady-state concentration of the specific substrate for the C4-reductase was calculated from the known equilibrium constants for the action of the epimerase with GDP-6-deoxy-4-keto- d - lyxo -heptose (see Figure ). The kinetic constants are provided in Table .…”

“…11 The predicted products for the HS:10A and HS:10B C4-reductases are GDP-6-deoxy-α-D-altro-heptose (3) and GDP-6-deoxy-β-L-galactoheptose (7), respectively, and thus, the HS:10B reductase is the one that was apparently expressed for the specific strain that was used for those investigations. This is interesting since the epimerase identified in the HS:10 serotype (UniProt id: F2X784) is likely restricted to the epimerization of only C3, 31 and thus, it is uncertain how C5 is epimerized for the ultimate formation of GDP-6-deoxy-β-L-galacto-heptose (7). For serotype HS:41, the reported heptose is 6-deoxy-D-altroheptose, 11 and the predicted products for the HS:41A and HS:41B C4-reductases are GDP-6-deoxy-D-altro-heptose and GDP-6-deoxy-L-galacto-heptose, respectively, and thus, the HS:41A C4-reductase was apparently expressed for the specific strain that was used for those investigations.…”

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

“…11,15,16 analysis of the DNA-sequenced serotypes of C. jejuni has demonstrated that there is a single common 4,6-dehydratase, 9 but two types of epimerases are utilized for the biosynthesis of the 6-deoxy-heptoses. 10 We have demonstrated that the largest class of epimerases (from serotypes HS:3, HS:4, HS:8, HS:10, HS:12, HS:23/36, HS:29, HS:33, and HS:41) is limited to the epimerization of C3, whereas the second-most populated class of epimerases (from serotypes HS:2, HS:15, HS:32, HS:42, and HS:63) catalyzes the racemization of C3 and C5. 10 For the eight C4-reductases that have been functionally characterized to date (serotypes HS:2, HS:3, HS:4, HS:15, HS:23/36, HS:33, HS:42, and HS:53), the epimerase in the gene cluster of that serotype is a C3/C5-epimerase if C5 is epimerized but is a C3-epimerase if C5 is not epimerized in the final 6-deoxy heptose product.…”

“…10 We have demonstrated that the largest class of epimerases (from serotypes HS:3, HS:4, HS:8, HS:10, HS:12, HS:23/36, HS:29, HS:33, and HS:41) is limited to the epimerization of C3, whereas the second-most populated class of epimerases (from serotypes HS:2, HS:15, HS:32, HS:42, and HS:63) catalyzes the racemization of C3 and C5. 10 For the eight C4-reductases that have been functionally characterized to date (serotypes HS:2, HS:3, HS:4, HS:15, HS:23/36, HS:33, HS:42, and HS:53), the epimerase in the gene cluster of that serotype is a C3/C5-epimerase if C5 is epimerized but is a C3-epimerase if C5 is not epimerized in the final 6-deoxy heptose product. 11 However, for some serotypes (namely, HS:8, HS:10, HS:29, HS:41, and HS:63), there are two nonredundant C4-reductases in the gene cluster.…”

“…Among the most common monosaccharide residues that are found in the structurally characterized capsular polysaccharides are heptoses. These relatively rare sugars include those that differ in stereochemistry at C2 through C6, in addition to deoxy versions at C3 and C6. − …”