Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance.

Lipopolysaccharide (LPS)1 is the major surface component of the outer membrane of Gram-negative bacteria. Its conserved element, lipid A, is the bioactive component predominantly responsible for many of the pathophysiological effects associated with Gram-negative bacterial infections (1, 2). Phosphate groups in lipid A and LPS result in a net negative charge on the bacterial outer surface. This overall negative charge is exploited by many multicellular organisms that produce cationic antimicrobial peptides (CAMPs) to defend themselves from bacterial invasion. CAMPs are small, amphipathic, positively charged peptides that constitute a conserved branch of the innate immune system (3–5). In Gram-negative bacteria, CAMPs bind through electrostatic interactions to the negatively charged groups of lipid A, causing membrane permeabilization and bacterial death (6–8). Gram-negative bacteria, including the human pathogens Salmonella typhimurium and Pseudomonas aeruginosa, can alter the structure of their cell wall to escape the host immune response (9–11). One mechanism that Gram-negative bacteria use to resist the bactericidal action of CAMPs is the addition of the positively charged sugar 4-amino-4-deoxy-L-arabinose (Ara4N) to lipid A (9, 11–13). This modification results in a less negatively charged bacterial outer membrane, decreasing the electrostatic binding of CAMPs to the bacterial cell wall. It has been clearly shown that lipid A modification with Ara4N is responsible for bacterial resistance to CAMPs and clinical antimicrobials such as polymyxin (14–16). Importantly, Ps. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients had lipid A modified by the addition of Ara4N (17). This modification facilitates the proliferation of bacteria in the CF lung (18). A clear understanding of the mechanism of Ara4N-lipid A biosynthesis is crucial for the development of combination therapies that combat bacterial resistance to CAMPs. The enzymes responsible for lipid A modification with Ara4N are located in the pmrHFIJKLM operon and the pmrE gene (pmr = polymyxin resistance) (19). They are under the direct control of the two-component system PmrA/PmrB (13, 20), which also regulates some other modifications of LPS including the addition of phosphoethanolamine to lipid A (21, 22). It has been shown that all of the gene products of the pmrHFIJKLM operon except pmrM are essential for the biosynthesis of Ara4N-lipid A and bacterial resistance to CAMPs and polymyxin (12). On the basis of sequence analyses, a pathway for the biosynthesis of Ara4N-lipid A has been proposed (23–26). In this pathway (Figure 1), UDP-Glc dehydrogenase (Ugd/PmrE) oxidizes UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA) in the presence of NAD+. UDP-GlcA is then oxidatively decarboxylated by the C-terminal domain of ArnA/PmrI (ArnA decarboxylase domain) to yield uridine 5′-diphospho-β-(L-threo-pentapyranosyl-4′′-ulose) (UDP-Ara4O). This intermediate is then transaminated by ArnB (PmrH) to produce UDP-4-aminoarabinose (UDP-Ara4N) in a thermodynamically unfavorable reaction (Keq = 0.1) (25). Raetz and coworkers have shown that ArnA can formylate UDP-Ara4N using N-10-formyltetrahydrofolate as the formyl donor (25), to yield uridine 5′-diphospho-β-(4-deoxy-4-formamido-L-arabinose) (UDP-Ara4NF) (27). However, no formylated products have been found modifying lipid A. Therefore, it has been proposed that UDP-Ara4N is transiently formylated by ArnA to compensate for the unfavorable equilibrium in the conversion of UDP-Ara4O to UDP-Ara4N. Preliminary data also obtained by Raetz and co-workers show the next enzyme in the pathway, ArnC (catalyzing the transfer of Ara4NF to undecaprenyl phosphate, thus preparing the molecule for translocation through the inner membrane), to be selective for UDP-Ara4NF (25). Very recent work by Raetz and co-workers shows that the transient formylation of UDP-Ara4N catalyzed by the N-terminal domain of ArnA is essential for both lipid A modification with Ara4N and polymyxin resistance (27). Figure 1 Part of the proposed pathway for the biosynthesis of Ara4N-lipid A (24, 25). The pathway starts with UDP-Glc being oxidized by Ugd/PmrE to form UDP-glucuronic acid. UDP-GlcA is then oxidatively decarboxylated by the ArnA decarboxylase domain (28) to yield ... ArnA (formerly PmrI) is a bifunctional 74 kDa protein (24, 25, 28). We have previously shown that the C-terminal region of Escherichia coli ArnA (amino acids 306–660) constitutes a separable domain fully responsible for the C-4′′ oxidation of UDP-GlcA to UDP-Ara4O (28). The N-terminal domain of ArnA (amino acids 1–300) displays similarity to enzymes involved in formyl transfer including glycinamide ribonucleotide formyltransferase, methionyl-tRNA formyltransferase, and the hydrolase domain of N-10-formyltet-rahydrofolate dehydrogenase (23, 25, 26). Here we show that the N-terminal domain of E. coli ArnA is, indeed, a separable domain responsible for the transformylation reaction. We designate it the ArnA transformylase domain and analyze its crystal structure refined to 1.7 A resolution.