Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116 ^†

Abstract

The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.

Investigation of the enzymes required for the biosynthesis of an unusual formylated sugar in the emerging human pathogen Helicobacter canadensis

“…25 Until now, those sugar ketoisomerases belonging to the cupin superfamily were thought to function as dimers. [26][27][28][29][30] Interestingly, the two H. canadensis subunits in the asymmetric unit did not display the typical dimeric quaternary structure. Upon inspection of the crystalline packing interactions, it became clear that the H. canadensis ketoisomerase adopted a tetrameric quaternary structure with D2 symmetry.…”

“…In some, however, His 51 is an asparagine and Tyr 35 is a phenylalanine. 29,30 To further verify that the protein encoded by HCAN_0198 is, indeed, a ketoisomerase, its kinetic parameters were determined via a continuous assay using dTDP-4-keto-6-deoxy-D-glucose as the substrate. A plot of initial velocity versus substrate concentration is provided in Figure 5, and the kinetic constants are listed in Table 1.…”

“…This value is similar to that observed for Tyl1a from Streptomyces fradiae (3.8 Â 10 3 M À1 s À1 ), 32 but lower for WlaRA and WlaRB from C. jejuni 81116 (2.9 Â 10 6 and 4.8 Â 10 5 M À1 s À1 , respectively). 30…”

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

“…The wlaRG gene (C. jejuni 81116, Penner serotype HS:6) was re-cloned using as a template the previously reported CJV-24 plasmid. 12 Primers were designed to incorporate NdeI and NotI restriction sites. The amplified wlaRG gene was digested with NdeI and NotI and ligated into pET28T-3g plasmid.…”

“…11 Shown in Scheme 1 are the proposed pathways leading to the formation of these 3,6-dideoxysugars. 12 As can be seen, both pathways initiate with the transfer of a dTMP moiety from dTTP to glucose-1-phosphate as catalyzed by a nucleotidylyltransferase (RmlA). In the second step, the C-6 0 carbon is reduced and the C-4 0 carbon is oxidized in an NAD-dependent reaction that is catalyzed by RmlB.…”