Functional and Structural Characterization of the UDP-Glucose Dehydrogenase Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni

Abstract

Campylobacter jejuni is a pathogenic organism that can cause campylobacteriosis in children and adults. Most commonly, campylobacter infection is brought on by consumption of raw or undercooked poultry, unsanitary drinking water, or pet feces. Surrounding the C. jejuni bacterium is a coat of sugar molecules known as the capsular polysaccharide (CPS). The capsular polysaccharide can be very diverse among the different strains of C. jejuni, and this diversity is considered important for evading the host immune system. Modifications to the CPS of C. jejuni NCTC 11168 include O-methylation, phosphoramidylation, and amidation of glucuronate with either serinol or ethanolamine. The enzymes responsible for amidation of glucuronate are currently unknown. In this study, Cj1441, an enzyme expressed from the CPS biosynthetic gene cluster in C. jejuni NCTC 11168, was shown to catalyze the oxidation of UDP-α-d-glucose into UDP-α-d-glucuronic acid with NAD+ as the cofactor. No amide products were found in an attempt to determine whether the putative thioester intermediate formed during the oxidation of UDP-glucose by Cj1441 could be captured in the presence of added amines. The three-dimensional crystal structure of Cj1441 was determined in the presence of NAD+ and UDP-glucose bound in the active site of the enzyme (Protein Data Bank entry 7KWS). A more thorough bioinformatic analysis of the CPS gene cluster suggests that the amidation activity is localized to the t-terminal half of Cj1438, a bifunctional enzyme that is currently annotated as a sugar transferase.

Discovery and Functional Characterization of a Clandestine ATP-Dependent Amidoligase in the Biosynthesis of the Capsular Polysaccharide from Campylobacter jejuni

“…We have shown that Cj1441 acts as a UDP-glucose 6dehydrogenase to make UDP-glucuronate (4). 14 This molecule is the most likely substrate for the glycosyltransferase that creates the glycosidic bond between D-glucuronate and the C2-hydroxyl from D-ribose at the growing end of the developing polysaccharide chain. The C-terminal domain of Cj1438 catalyzes amide bond formation between the Dglucuronate moiety and (S)-serinol phosphate to make the corresponding glucuronamide phosphate.…”

“…The enzyme was crystallized and the protein structure determined to a resolution of 2.1 Å in the presence of UDP-glucose and NAD + . 14 ethanolamine phosphate (8), whereas Cj1437 is a PLPdependent transaminase that catalyzes the stereospecific transamination of dihydroxyacetone phosphate to (S)-serinol phosphate (7). 15 The gene clusters for the biosynthesis of the CPSs made by C. jejuni NCTC 11168 (HS:2) and C. jejuni RM1285 (HS:19) are shown in Figure S1.…”

“…Previously, we investigated the roles of the enzymes Cj1441, Cj1436, and Cj1437 in the biosynthetic pathway for the assembly of the CPS from C. jejuni NCTC 11168. , Cj1441 is a UDP-glucose 6-dehydrogenase that catalyzes the dual oxidation of UDP- d -glucose to UDP- d -glucuronate ( 4 ). The enzyme was crystallized and the protein structure determined to a resolution of 2.1 Å in the presence of UDP-glucose and NAD + .…”

Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni

“…8,20−25 More recently, we have shown that Cj1441 is required for the conversion of uridine diphosphate (UDP)-glucose to UDP-glucuronate and that this enzyme is unable to catalyze the formation of an amide bond via the attack of an amine substrate with the putative thioester intermediate formed during the oxidation of UDP-glucose. 26 The enzymes required for the biosynthesis of the two amines (serinol and ethanolamine) found in the HS:2 capsule are currently unknown.…”

“…jejuni NCTC 11168 is composed of 35 genes, as illustrated in Figure S1 . Previous investigations have functionally characterized the genes necessary for the biosynthesis of the d -glycero- l -gluco-heptose moiety and modification (Cj1152, Cj1423, Cj1424, Cj1425, Cj1427, Cj1428, and Cj1430). − In addition, the enzymes Cj1415, Cj1416, Cj1417, and Cj1418 have been shown to be required for the biosynthesis of the phosphoramidate modification, and gene knockout experiments have identified the enzymes (Cj1422 and Cj1421) required for the transfer of the phosphoramidate modifications to specific sugar receptors. ,− More recently, we have shown that Cj1441 is required for the conversion of uridine diphosphate (UDP)-glucose to UDP-glucuronate and that this enzyme is unable to catalyze the formation of an amide bond via the attack of an amine substrate with the putative thioester intermediate formed during the oxidation of UDP-glucose . The enzymes required for the biosynthesis of the two amines (serinol and ethanolamine) found in the HS:2 capsule are currently unknown.…”