Origin of Lipid A Species Modified with 4-Amino-4-deoxy-l-arabinose in Polymyxin-resistant Mutants of Escherichia coli

Abstract

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-(␤-L-threo-pentapyranosyl-4؆-ulose diphosphate), the intermediate that is synthesized by ArnA from UDPglucuronic acid. A 1.7-mg sample of the putative UDP-LAra4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1 H and 13 C 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 (ϳ0.1). 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.

An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli

“…The forward typhimurium, biosynthesis of the L-Ara4N moiety begins with oxidation of UDP-glucose to UDP-glucuronic acid (2). Next, the C-terminal domain of ArnA catalyzes the NAD ϩ -dependent oxidative decarboxylation of UDP-glucuronic acid to yield an unusual UDP-4-ketopentose (30,32), which is converted by the transaminase ArnB to UDP-␤-L-Ara4N (29). Subsequently, the N-terminal domain of ArnA uses N-10-formyltetrahydrofolate to N-formylate UDP-␤-L-Ara4N (28,30,32).…”

Crystal Structure ofEscherichia coliArnA (PmrI) Decarboxylase Domain. A Key Enzyme for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance,

“…In addition to their function as a key member of the innate immune system, CAMPs represent an important class of clinical antimicrobials. They have both intrinsic bactericidal activity and appear to enhance the activity of other antibiotics, presumably by facilitating their entry into the microbe (3,10,11 21). The N-terminal domain (residues 1-313) is similar in sequence to other enzymes involved in formyl transfer.…”

“…All of these gene products except pmrM are essential for the biosynthesis of Ara4N-lipid A and for resistance to CAMPs (18). In vitro studies by Raetz and co-workers have shown a pathway for the biosynthesis of UDP-L-Ara4N from UDP-glucose (UDP-Glc) (19)(20)(21). The pathway begins with the oxidation of UDP-Glc to UDP-glucuronic acid (UDP-GlcA) catalyzed by the well-characterized UDP-Glc dehydrogenase (PmrE/Ugd) ( Figure 1).…”

“…UDP-GlcA is then oxidatively decarboxylated by ArnA (PmrI) to yield UDP-4-keto-arabinose (UDPAra4O), which in turn is transaminated to produce UDP-4-amino-arabinose (UDP-Ara4N) in a reaction catalyzed by ArnB (PmrH). On the basis of sequence similarity to enzymes with known activities, additional gene products of the pmrHFIJKLM operon have been proposed to catalyze the transfer of Ara4N from the UDP intermediate to lipid A (19,(21)(22)(23)(24). The enzymes in this pathway are potential targets for antibacterial drug design.…”

Expression Cloning of Three Rhizobium leguminosarum Lipopolysaccharide Core Galacturonosyltransferases

“…Based on its homology to yeast dolichyl-phosphate mannose synthase, E. coli PmrF was postulated to be involved in the synthesis of the lipid-linked L-Ara4N donor (58). In fact, PmrF (ArnC) catalyzes the selective transfer of L-Ara-formyl-4N from UDP-N-formyl-4-amino-4-deoxy-L-arabinose to undecaprenyl phosphate, generating a lipid precursor that is immediately deformylated by ArnD (59,60). The final lipid donor, undecaprenyl phosphate-␣-L-Ara4N, isolated in milligram quantities from the lipid fraction of polymyxin-resistant mutants, is well characterized (35).…”