Multi-dimensional NMR structural analyses of purified Lipid X and Lipid A (endotoxin)

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Polymyxin-resistant mutants of Escherichia coli andPolyisoprene-linked sugars function as donor substrates for many types of glycosyltransferases (1). In eubacteria, undecaprenyl moieties (1, 2) serve as lipid carriers for sugar residues that are transferred to acceptors located outside of the cytoplasm, where sugar nucleotides are not available. Undecaprenyl diphosphate-sugars (generally oligosaccharides) are precursors for polymerization of O-antigen (3-6), enterobacterial common antigen (7,8), and peptidoglycan (1, 9 -11). Undecaprenyl phosphate-sugars (typically monosaccharides) are thought to be donors for processes that include bacteriophagemediated O-antigen conversion (12, 13), glycosylation of teichoic acids (14 -17), and biosynthesis of mycobacterial lipoglycans (18 -21). In eucaryotic cells, the structurally related dolichyl phosphate-sugars and dolichyl diphosphate-sugars (1, 22-24) play important roles in various stages of protein glycosylation and in the assembly of phosphatidylinositol-linked glycans.As demonstrated in the preceding article (25), a membranebound donor, proposed to be undecaprenyl phosphate-␣-LAra4N 1 (Fig. 1) 2 based upon bioinformatic considerations (26, 27), is required for the modification of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) units in polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium. A novel L-Ara4N transferase, encoded by arnT (previously designated orf5 or pmrK) (28, 29), catalyzes L-Ara4N transfer to lipid A-like molecules in vitro when membranes of polymyxin-resistant mutants are employed as the source of the L-Ara4N donor (25). The formation of L-Ara4N and its transfer to lipid A are induced by activation of the transcription factor PmrA, which may occur by mutation (30 -32), by activation of PhoP (33, 34), or by exposure of cells to mildly acidic pH, ferric ions, or metavanadate (26,35,36). Attachment of the positively charged L-Ara4N moiety to lipid A is critical for resistance to the antibiotic polymyxin and to certain cationic antimicrobial peptides present inside phagocytic cells (37,38).We now report the purification and structural characterization of a novel, minor lipid that accumulates in polymyxinresistant mutants of E. coli and S. typhimurium. The purified lipid functions as a donor of L-Ara4N residues in the ArnTcatalyzed modification of lipid A in vitro. MALDI/TOF mass spectrometry and high resolution NMR spectroscopy strongly * This work was supported in part by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM54882 (to R. J. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Radiolabeled Substrates-[4ј-mentioning

confidence: 99%

Section: Characterization Of the Purified Lipid By Two-dimensionalmentioning

confidence: 99%

See 1 more Smart Citation

Accumulation of a Polyisoprene-linked Amino Sugar in Polymyxin-resistant Salmonella typhimurium andEscherichia coli

Trent¹,

Ribeiro²,

Doerrler³

et al. 2001

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“…31 P spectra at 202 MHz were obtained with a 12 kHz SW, 59°pulse flip angle (13 s), 2.4-s acquisition time, and 1.6-s RD, and digitized using 25000 points to obtain a digital resolution of 0.97 Hz/pt. Selective inverse decoupling difference spectroscopy (43,44) was implemented by detecting the 1 H NMR spectrum at high digitization while selectively exciting a phosphorus signal with 1 s of low-power (estimated 0.2 mW, ϳ4 db on the Unity 500 spectrometer) continuous wave on-and off-resonance irradiation during the acquisition period. To improve subtraction in the generation of one-dimensional difference 31 P-decoupled 1 H NMR spectra, the samples were thermally equilibrated in the magnet for over 1 h before recording data, and the 1 H difference spectra were obtained in an interleaved manner with 4 scans accumulated for each free induction decay and looping around n times to achieve a good signal-to noise ratio with 4n scans per free induction decay.…”

Section: Fig 2 Biosynthesis Of Udp-l-ara4n From Udp-glca In Polymyxmentioning

confidence: 99%

Oxidative Decarboxylation of UDP-Glucuronic Acid in Extracts of Polymyxin-resistant Escherichia coli

Breazeale¹,

Ribeiro²,

Raetz³

2002

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Addition of the 4-amino-4-deoxy-L-arabinose (L-Ara4NPmrI (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-(␤-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.

“…The structural details of lipid A substitution patterns could not be deduced unequivocally from such preparations. Recent improvements in lipid A purification methods, in conjunction with new procedures for high resolution NMR spectroscopy and mass spectrometry (7,19,24), now provide an opportunity to analyze the lipid A modifications of S. typhimurium in a more definitive manner. This effort is essential as a prelude to enzymatic studies of lipid A modifications.…”

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confidence: 99%

Lipid A Modifications in Polymyxin-resistant Salmonella typhimurium

Zhou

Ribeiro

Lin

et al. 2001

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128

Salmonella typhimurium and related organisms synthesize lipid A by the same pathway as Escherichia coli K-12 (1, 2), but they usually modify the final product with additional covalent appendages (Fig. 1A), such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) 1 (3-7), phosphoethanolamine (pEtN) (4 -6), (S)-2-hydroxymyristate (8, 9), and palmitate (5, 6, 10 -12). Different combinations of these substituents account for the remarkable heterogeneity of lipid A molecules found in S. typhimurium.The biosynthesis of lipid A modifications is under the control of the PhoP/PhoQ and the PmrA/PmrB two-component signaling systems (13-15). Addition of the L-Ara4N unit is required for resistance to polymyxin (16 -18). Incorporation of the palmitoyl chain confers resistance to certain cationic anti-microbial peptides (11). Modification of lipid A with L-Ara4N, pEtN, and/or palmitate may also occur in E. coli K-12, but only under special circumstances, as in polymyxin-resistant (pmrA constitutive) mutants (17) or in wild type cells exposed to ammonium metavanadate (7,19).With the exception of PagP, the outer membrane enzyme that incorporates palmitate (12, 20), the enzymes responsible for the covalent modifications of lipid A have not been identified. The L-Ara4N residue is attached primarily to the 4Ј-phosphate group of lipid A in wild type S. typhimurium or in metavanadate-treated E. coli, whereas pEtN is usually attached to the 1-phosphate (19). However, in S. typhimurium mutants defective in Kdo biosynthesis, lipid A precursors accumulate in which L-Ara4N is linked exclusively to the 1-phosphate, and pEtN is attached mainly to the 4Ј-phosphate (5,6,19). The enzymatic pathways that account for these interesting and complex structural anomalies are unknown.An important clue to the origin of the L-Ara4N moiety has emerged from the discovery of the pmrE and pmrF genes, which are required for the maintenance of polymyxin resistance and the biosynthesis of L-Ara4N-modified lipid A (18). The pmrE (ugd) gene encodes UDP-glucose dehydrogenase (18), suggesting that L-Ara4N is derived from UDP-glucuronic acid. The pmrF gene encodes a homologue of yeast dolichyl phosphate-mannose synthase and is part of an operon (18) that includes additional open reading frames hypothesized to encode other putative enzymes required for L-Ara4N biosynthesis and attachment to lipid A (7, 21). The operon is regulated directly by PmrA (18), which in turn may be activated by PhoP/PhoQ, low pH, or ferric ions (14,22,23). So far, no in vitro assays have been developed to validate the functions of the proteins encoded by the pmrF operon.Because of its heterogeneity (Fig. 1A), S. typhimurium lipid * This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H.R.), AI-30479 (to S. I. M.), and GM54882 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.** To w...