Pantoea ananatis is a Gram-negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non-pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4-formamido-4,6-dideoxy-Dglucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6-dehydratase, an aminotransferase, and an N-formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N-formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis-Menten kinetics with dTDP-4-amino-4,6-dideoxy-D-glucose as the substrate and N 10 -formyltetrahydrofolate as the carbon source. In addition, the X-ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N-formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N-formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non-pathogenic apparently do not contain these genes.

Psychrobacter cryohalolentis K5 T is a Gram-negative bacterium first isolated from Siberian permafrost in 2006. It has a complex O-antigen containing L-rhamnose, D-galactose, two diacetamido-sugars, and one triacetamido-sugar. The biosynthetic pathway for one of the diacetamido-sugars, namely 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid, is presently unknown. Utilizing the published genome sequence of P. cryohalolentis K5 T , we hypothesized that the genes designated Pcryo_0613, Pcryo_0614, Pcryo_0616, and Pcryo_0615 encode for a uridine dinucleotide (UDP)-N-acetyl-D-glucosamine 6-dehydrogenase, an nicotinamide adenine dinucleotide (oxidized) (NAD + )-dependent dehydrogenase, a pyridoxal 5 0 -phosphate (PLP)-dependent aminotransferase, and an N-acetyltransferase, respectively, activities of which would be required for the biosynthesis of this unusual carbohydrate. Here we present the cloning, overexpression, and purification of these hypothetical proteins. Kinetic data on the enzymes encoded by Pcryo_0613, Pcryo_0614, and Pcryo_0615 confirmed their postulated biochemical activities. In addition, the high-resolution X-ray structures of both the internal and external aldimine forms of the aminotransferase were determined to 1.25 and 1.0 Å, respectively. Finally, the three-dimensional architecture of the N-acetyltransferase in complex with its substrate and coenzyme A was solved to 1.8 Å resolution. Strikingly, the N-acetyltransferase was shown to adopt a new motif for UDP-sugar binding. The data presented herein provide additional insight into sugar biosynthesis in Gram-negative bacteria.Abbreviations: CoA, coenzyme A; HEPES, N-2-hydroxyethylpiperazie-N 0 -2-ethanesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N 0 -3-propanesulfonic acid; HPLC, high-performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; NAD + , nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NDP, nucleotide diphosphate; Ni-NTA, nickel-nitrilotriacetic acid; PLP, pyridoxal 5 0 -phosphate; rTEV, recombinant tobacco etch virus; Tris, tris-(hydroxymethyl)aminomethane; UDP, uridine dinucleotide; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine.

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