Publications

• Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose.

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

  In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C ... >> More

  In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety. << Less

  J. Biol. Chem. 278:24731-24739(2003) [PubMed] [EuropePMC]

• Structural studies of Salmonella typhimurium ArnB (PmrH) aminotransferase: a 4-amino-4-deoxy-L-arabinose lipopolysaccharide-modifying enzyme.

  Noland B.W., Newman J.M., Hendle J., Badger J., Christopher J.A., Tresser J., Buchanan M.D., Wright T.A., Rutter M.E., Sanderson W.E., Mueller-Dieckmann H.-J., Gajiwala K.S., Buchanan S.G.

  Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a ... >> More

  Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site. << Less

  Structure 10:1569-1580(2002) [PubMed] [EuropePMC]

• Structural basis for substrate specificity in ArnB. A key enzyme in the polymyxin resistance pathway of Gram-negative bacteria.

  Lee M., Sousa M.C.

  Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their me ... >> More

  Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas , have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar-nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the (4)C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients. << Less

  Biochemistry 53:796-805(2014) [PubMed] [EuropePMC]