Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N⁵‐formyltetrahydrofolate and UDP‐Ara4N

Abstract

ArnA from Escherichia coli is a key enzyme involved in the formation of 4-amino-4-deoxy-L-arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram-negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N-and C-terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N 10 -formyltetrahydrofolate. Here we describe the structure of the isolated N-terminal domain of ArnA in complex with its UDP-sugar substrate and N 5 -formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.Keywords: N-formyltransferase; 4-amino-4-deoxy-L-arabinose; N 10 -formyltetrahydrofolate; lipopolysaccharide; resistance to cationic antimicrobial peptides Abbreviations: Ara4N, 4-amino-4-deoxy-L-arabinopyranose; HEPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid; HPLC, high performance liquid chromatography; NAD 1 , nicotinamide adenine dinucleotide; Ni-NTA, nickel nitrilotriacetic acid; TEV, tobacco etch virus; dTMP, thymidine monophosphate; Tris, tris-(hydroxymethyl)aminomethane; UMP, uridine monophosphate.The authors have no competing financial interests.X-ray coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, N. J. (accession no. 5J63).Broader statement: ArnA from Escherichia coli is a key enzyme involved in the formation of 4-amino-4-deoxy-L-arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram-negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Here we present a model for the enzyme in the presence of its UDP-sugar substrate. This model provides a new scaffold for structure-based drug design.

Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis

“…Whereas these nucleotide-linked sugars are not commercially available, they have been enzymatically synthesized in our laboratory. 8–12,14,25 From this list, the only nucleotide-linked sugar that served as a substrate for Rv3404c was dTDP-Qui4N. Specifically, the mass spectrum for dTDP-Qui4N shows a peak having a m/z = 545.9.…”

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

“…Given our long‐standing interest in unusual sugar biosynthesis, and in particular on N ‐formylated sugars, we utilized a simple bioinformatics analysis to determine whether any strains of P. ananatis contained the genes required for the production of such carbohydrates. Briefly, we performed a BLAST® search to discover gene sequences that were similar to those encoding enzymes previously investigated in the laboratory . The vast majorities of “hits” were annotated as l ‐methionyl‐tRNA N ‐formyltransferases.…”

Modeling of interactions between functional domains of ALDH1L1

“…The recently solved structure of this domain complexed with UDP-Ara4N also shows the formyltransferase fold (32). As with WlaRD, the β2-α2 loop (H32–G43) in the N-terminal lobe, and the α–β loop in the C-terminal lobe (V224–A231) block the bottom of the binding cleft.…”