Oxidation and transamination of the 3-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans -: Role in the formation of lipid a molecules with four amide-linked acyl chains

Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a beta-1',6-linked glucosamine disaccharide backbone. However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-D-glucopyranose. We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino2,3-dideoxy-D-glucopyranose moiety. The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A. ferrooxidans. Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD(+)-dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP2-acetamido-3-amino2,3-dideoxy-alpha-D-glucopyranose (UDP-GlcNAc3N). Both gnnA and gnnB were cloned and expressed in E. coli using pET23c+. In the presence of L-glutamate and NAD(+), both proteins were required for the conversion of [alpha-P-32]UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be [alpha-P-32]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, similar to0.4 mg of the presumptive UDP-GlcNAc3N was synthesized. The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH2. As shown in the accompanying papers ( Sweet, C. R., Williams, A. H., Karbarz, M. J., Werts, C., Kalb, S. R., Cotter, R. J., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25411-25419; Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. ( 2004) J. Biol. Chem. 279, 25420-25429), UDP-GlcNAc3N is selectively acylated by LpxAs of A. ferrooxidans, L. interrogans, and M. loti. UDP-GlcNAc3N may be useful as a substrate analog for diverse enzymes that utilize UDP-GlcNAc.