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

• Published in: The Journal of biological chemistry Vol. 279; no. 24; pp. 25400 - 25410
• Main Authors: Sweet, Charles R, Ribeiro, Anthony A, Raetz, Christian R H
• Format: Journal Article
• Language: English
• Published: United States 11-06-2004
• Subjects: Acidithiobacillus - enzymology; Acidithiobacillus - genetics; Acidithiobacillus ferrooxidans; Amino Acid Sequence; Escherichia coli; Legionella pneumophila; Leptospira interrogans; Lipid A - biosynthesis; Lipid A - chemistry; Magnetic Resonance Spectroscopy; Mesorhizobium loti; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases - genetics; Oxidoreductases - physiology; Transaminases - genetics; Transaminases - physiology; Uridine Diphosphate N-Acetylglucosamine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M400596200

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-diamino-2,3-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 UDP 2-acetamido-3-amino-2,3-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-(32)P]UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be [alpha-(32)P]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, approximately 0.4 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 NH(2). As shown in the accompanying papers, 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.