Many
strains of Campylobacter jejuni display modified
heptose residues in their capsular polysaccharides (CPS). The precursor
heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a
variety of modifications of the sugar moiety have been observed. These
modifications include the generation of 6-deoxy derivatives and alterations
of the stereochemistry at C3–C6. Previous work has focused
on the enzymes responsible for the generation of the 6-deoxy derivatives
and those involved in altering the stereochemistry at C3 and C5. However,
the generation of the 6-hydroxyl heptose residues remains uncertain
due to the lack of a specific enzyme to catalyze the initial oxidation
at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously
reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound
NADH, thus hindering catalysis of oxidation reactions. However, addition
of a co-substrate, α-ketoglutarate, converts the bound NADH
to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal
structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the
NAD(H) cofactor is ideally positioned to catalyze the oxidation at
C4 of the sugar substrate. Additionally, the overall fold of the Cj1427
subunit places it into the well-defined short-chain dehydrogenase/reductase
superfamily. The observed quaternary structure of the tetrameric enzyme,
however, is highly unusual for members of this superfamily.
N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-D-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-D-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handedβ-helix superfamily (LβH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-D-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens
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