Publications

• A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-amino-4-deoxy-L-arabinose: identification and function of UDP-4-deoxy-4-formamido-L-arabinose.

  Breazeale S.D., Ribeiro A.A., McClerren A.L., Raetz C.R.H.

  Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidat ... >> More

  Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid to yield the UDP-4''-ketopentose, uridine 5'-diphospho-beta-(L-threo-pentapyranosyl-4''-ulose), which is converted by ArnB to UDP-beta-(L-Ara4N). E. coli ArnA is a bi-functional enzyme with a molecular mass of approximately 74 kDa. The oxidative decarboxylation of UDP-glucuronic acid is catalyzed by the 345-residue C-terminal domain of ArnA. The latter shows sequence similarity to enzymes that oxidize the C-4'' position of sugar nucleotides, like UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase. We now show that the 304-residue N-terminal domain catalyzes the N-10-formyltetrahydrofolate-dependent formylation of the 4''-amine of UDP-L-Ara4N, generating the novel sugar nucleotide, uridine 5'-diphospho-beta-(4-deoxy-4-formamido-L-arabinose). The N-terminal domain is highly homologous to methionyl-tRNA(f)Met formyltransferase. The structure of the formylated sugar nucleotide generated in vitro by ArnA was validated by 1H and 13C NMR spectroscopy. The two domains of ArnA were expressed independently as active proteins in E. coli. Both were required for maintenance of polymyxin resistance and L-Ara4N modification of lipid A. We conclude that N-formylation of UDP-L-Ara4N is an obligatory step in the biosynthesis of L-Ara4N-modified lipid A in polymyxin-resistant mutants. We further demonstrate that only the formylated sugar nucleotide is converted in vitro to an undecaprenyl phosphate-linked form by the enzyme ArnC. Because the L-Ara4N unit attached to lipid A is not derivatized with a formyl group, we postulate the existence of a deformylase, acting later in the pathway. << Less

  J. Biol. Chem. 280:14154-14167(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• An undecaprenyl phosphate-aminoarabinose flippase required for polymyxin resistance in Escherichia coli.

  Yan A., Guan Z., Raetz C.R.H.

  Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the Pm ... >> More

  Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA(c) parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. Lipid A was no longer modified with L-Ara4N, even though the levels of the lipid-linked donor of the L-Ara4N moiety, undecaprenyl phosphate-alpha-L-Ara4N, were not reduced in the mutants. However, the undecaprenyl phosphate-alpha-L-Ara4N present in the mutants was less concentrated on the periplasmic surface of the inner membrane, as judged by 4-5-fold reduced labeling with the inner membrane-impermeable amine reagent N-hydroxysulfosuccin-imidobiotin. In an arnT mutant of the same pmrA(c) parent, which lacks the enzyme that transfers the L-Ara4N unit to lipid A but retains the same high levels of undecaprenyl phosphate-alpha-L-Ara4N as the parent, N-hydroxysulfosuccinimidobiotin labeling was not reduced. These results implicate pmrL and pmrM, but not arnT, in transporting undecaprenyl phosphate-alpha-L-Ara4N across the inner membrane. PmrM and PmrL, now renamed ArnE and ArnF because of their involvement in L-Ara4N modification of lipid A, may be subunits of an undecaprenyl phosphate-alpha-L-Ara4N flippase. << Less

  J. Biol. Chem. 282:36077-36089(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.