Reaction Mechanism and Three-Dimensional Structure of GDP-d-glycero-α-d-manno-heptose 4,6-Dehydratase from Campylobacter jejuni

Abstract

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD+ in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89–98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.

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

“…For all serotypes sequenced to date, there appears to be a single type of 4,6-dehydratase that is common throughout. , The global sequence identity for the entire set of dehydratases from 19 C. jejuni serotypes ranges from 89 to 98% .…”

“…For all serotypes sequenced to date, there appears to be a single type of 4,6-dehydratase that is common throughout. , The global sequence identity for the entire set of dehydratases from 19 C. jejuni serotypes ranges from 89 to 98% . With the C3/C5-epimerases, there appears to be three different clusters of enzymes with different stereochemical outcomes.…”

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

“…DNA sequencing was conducted by Eton Biosciences, Inc. GDP-Dglycero-α-D-manno-heptose (1) was enzymatically synthesized and purified as described previously. 9 Plasmid Construction. The genes encoding the C4reductases from different serotypes of C. jejuni were codonoptimized for expression in E. coli and ordered from either Twist Biosciences (San Francisco, CA) or GenScript (Piscataway, NJ) in a pET28a (+) expression vector with an Nterminal hexa-histidine tag.…”

“…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. − …”

“…It is currently thought that all of the heptoses found in the capsular polysaccharides in C. jejuni originate from GDP- d - glycero - d - manno -heptose ( 1 ). ,− For the biosynthesis of the 6-deoxy-heptoses, the generally accepted biosynthetic pathway involves the successive activities of a 4,6-dehydratase, a C3- or C3/C5-epimerase, and a C4-reductase, as shown in Figure . − ,− From this biosynthetic pathway, eight different 6-deoxyheptoses are possible (three stereocenters at C3, C4, and C5), and we and Creuzenet’s group have characterized the C4-reductases from multiple serotypes that can synthesize six of the eight possible products (Figure ). ,, A bioinformatic analysis of the DNA-sequenced serotypes of C.…”