Structure of a sugar N-formyltransferase from Campylobacter jejuni.

The outer membranes of Gram-negative bacteria are characterized by a complex lipoglycan referred to as the lipopolysaccharide, or LPS. It can be envisioned in terms of three components: (1) lipid A, which serves to anchor the LPS to the outer membrane, (2) the core oligosaccharide, which contains sugars such as glucose, galactose, and N-acetylglucosamine as well as some seven-carbon sugars, and (3) the O-antigen or O-polysaccharide, which extends farthest away from the bacterium. Several mucosal pathogens, such as Neisseria, Haemophilus, and Campylobacter spp., lack O-polysaccharide repeating units and display considerable structural diversity in their outer core glyans.1 These low molecular weight LPS variants are referred to as lipooligosaccharides (LOS). The O-polysaccharides and LOS outer cores often contain unusual sugars such as colitose, paratose, or tyvelose, among others.2 Indeed, more than 100 different di-, tri-, and tetradeoxysugars have been isolated from prokaryotic sources thus far.3 Whereas it has been established that lipid A is a major contributor to the toxicity of Gram-negative bacterial infections, the biological role of the O-polysaccharide is less well understood. Recent studies suggest that it is important for the effective colonization of host tissues and for resistance to complement-mediated killing.4 Through the efforts of numerous laboratories, a better understanding of the manner in which unusual di-, tri-, and tetradeoxysugars are biochemically synthesized has been obtained.5 Many are constructed around the nucleotide-linked 3,6-dideoxyhexoses, which are further modified by acetylation, amination, epimerization, and C-, N-, and O-methylation reactions. At present, there is a substantial body of literature describing these types of enzymatic modifications. There is still a paucity of structural and kinetic data on N-formyltransferases, however, that function specifically on nucleotide-linked sugars. This is somewhat surprising given that the existence of N-formylated sugars was reported back in 1985.6 Thus far, only two biochemical studies have been described on sugar N-formyltransferases. The first was on ArnA from Escherichia coli, which catalyzes the conversion of UDP-4-amino-4-deoxy-L-arabinose to UDP-4-formamido-4-deoxy-L-arabinose.7 ArnA is a bifunctional protein, with the N-terminal domain functioning as the N-formyltransferase and the C-terminal domain catalyzing an oxidative decarboxylation reaction. The second enzyme investigated, VioF from Providencia alcalifaciens O3, catalyzes the conversion of dTDP-4-amino-4,6-dideoxy-D-glucose to dTDP-4-formamido-4,6-dideoxy-D-glucose.8 Both ArnA and VioF use N10-formyltetrahydrofolate (N10-formyl-THF) as the formyl donor. A crystal structure of the N-terminal domain of ArnA was determined in the presence of N5-formyltetrahydrofolate (N5-formyl-THF) and UMP, but details concerning the active site architecture were limited due to the absence of a bound nucleotide-linked sugar.9 Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E LOS biosynthesis locus containing 19 genes. This locus includes 11 homologues of glycosyltransferases, one lipid A acyltransferase, and 7 genes involved in either the biosynthesis of dideoxyhexosamine residues (ddHexN) or in their modifications.10 The basic composition of the C. jejuni LOS outer core has been proposed through mass spectrometry analysis, and it has been suggested that it contains two ddHexN residues that can be either acetylated or formylated.10 However, the complexity and heterogeneity of the glycans produced by C. jejuni 81116 prevented the complete structural determination of the LOS outer core by NMR. In addition, two other polysaccharides are produced by C. jejuni 81116: polysaccharide A, which contains glucose, glucuronic acid, and mannose, and polysaccharide B, which contains glucose, galactose, and N-acetylglucosamine.11 Recently, we have shown that the hypothetical protein C8J_1081 from C. jejuni 81116 catalyzes the transfer of a formyl group to either dTDP-3-amino-3,6-dideoxy-D-glucose (dTDP-Qui3N) or dTDP-3-amino-3,6-dideoxy-D-galactose (dTDP-Fuc3N) as indicated in Scheme 1 (the protonation states of these substrates, when bound in the enzyme’s active site, are not known). Here we report a combined structural and functional investigation of this enzyme, hereafter referred to as WlaRD. For this analysis, seven structures were determined to a resolution of 1.9 A or better: complexes of the enzyme with N5-formyl-THF and dTDP, dTDP-Qui3N, dTDP-Fuc3N, or dTDP-3,6-dideoxy-3-formamido-D-galactose (dTDP-Fuc3NFo), complexes of the enzyme with N10-formyl-THF and dTDP or dTDP-glucose, and a complex of WlaRD with tetrahydrofolate (THF) and dTDP-3,6-dideoxy-3-formamido-D-glucose (dTDP-Qui3NFo). On the basis of these structures, site-directed mutant proteins were constructed and their kinetic parameters measured. Taken together, these studies provide, for the first time, a glimpse of a fully loaded sugar N-formyltransferase active site and reveal how enzymes in this class bind N10-formyl-THF. Scheme 1