Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose

J Biol Chem. 2002 Jan 25;277(4):2886-96. doi: 10.1074/jbc.M109377200. Epub 2001 Nov 8.

Abstract

Addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system. The sequences of the products of the Salmonella typhimurium pmrE and pmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for l-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R. J., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 18503-18514). We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of [alpha-(32)P]UDP-glucuronic acid, followed by transamination to generate [alpha-(32)P]UDP-l-Ara4N, when NAD and glutamate are added as co-substrates. In addition, the [alpha-(32)P]UDP-l-Ara4N is formylated when N-10-formyltetrahydrofolate is included. These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon. PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate). A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone. ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Sugars / chemistry*
  • Carbohydrate Sequence
  • Cell Membrane / metabolism
  • Cell-Free System
  • Cytoplasm / metabolism
  • Drug Resistance*
  • Escherichia coli / metabolism*
  • Glutamic Acid / pharmacology
  • Lipid A / metabolism*
  • Magnetic Resonance Spectroscopy
  • Models, Chemical
  • Molecular Sequence Data
  • Mutation
  • NAD / pharmacology
  • Oxygen / metabolism*
  • Polymyxins / pharmacology*
  • Protein Binding
  • Protein Structure, Tertiary
  • Time Factors
  • Uridine Diphosphate Glucose / metabolism
  • Uridine Diphosphate Glucuronic Acid / metabolism*

Substances

  • Amino Sugars
  • Lipid A
  • Polymyxins
  • NAD
  • Uridine Diphosphate Glucuronic Acid
  • 4-amino-4-deoxyarabinose
  • Glutamic Acid
  • Oxygen
  • Uridine Diphosphate Glucose

Associated data

  • GENBANK/AY057445