Structure–function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways

Abstract

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6- d eoxy- D - a ltro - h eptose and GDP-6-O M e- L - g luco - h eptose in Campylobacter jejuni . However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

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

“…In addition to these differences, there are five residues near the two critical active site residues (His-67 and Tyr-134) that are differentially conserved between the two groups of enzymes. 12,15 These residues include Ala-76, Ala-122, Glu-128, Ser/Thr-129, and Cys-136 for the C3epimerases and Val-76, Ser-122, Lys-128, Glu-129, and Leu-136 for the C3/C5-epimerases (Figure 6).…”

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

“…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.…”

Biosynthesis of d-glycero-l-gluco-Heptose in the Capsular Polysaccharides of Campylobacter jejuni

“…Whereas NADPH was specifically included in the crystallization trials for Cj1430, the presented data in Barnawi et al do not specifically address the oxidation state of the cofactor in the crystals. 29…”

“…Recently, Naismith and Creuzenet reported the three-dimensional structures of Cj1428 (aka MlghC) in the presence of NADP (PDB id: 7ANC) and Cj1430 (aka MlghB) in the presence of GDP-mannose (PDB id: 7AN4). These two proteins were functionally characterized using GDP-mannose and GDP-6-deoxy- manno -heptose based substrates in contrast to the more physiological GDP- d - glycer o- d - manno -heptose based substrates employed in this investigation. Nevertheless, that investigation clearly demonstrated that MlghB catalyzes the epimerization at C3 and C5 of the 4-keto-mannose moiety of the substrate and that MlghC catalyzes the reduction of the corresponding reaction product generating GDP-6-deoxy- L - gluco -heptose.…”

“…With respect to the models of Cj1428 determined in the presence of NADPH and MlghC solved in the presence of NADP + , they are basically identical (α-carbons superimpose with a root-mean-square deviation of 0.2 Å). Whereas NADPH was specifically included in the crystallization trials for Cj1430, the presented data in Barnawi et al do not specifically address the oxidation state of the cofactor in the crystals …”