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

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 similar to 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 H-1 and C-13 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.