Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Lipid modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed2006

Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells.

Posttranslational modifications, including phosphorylation, ubiquitination and lipid modifications, provide proteins with additional functions and regulation beyond genomic information. Palmitoylation is a reversible lipid modification with palmitic acid that plays critical roles in protein trafficking and function. However, the enzymes that mediate palmitoyl acyl transferase (PAT) have been elusive. Recent genetic analysis in yeast revealed that members of cysteine-rich DHHC domain containing proteins (DHHC proteins) mediate palmitoylation. In mammalian genomes, 23 DHHC proteins are predicted raising the possibility of a large family of PAT enzymes. Here, we describe a systematic method to examine which of the DHHC family members is responsible for palmitoylation of a substrate.

Acylation↗
PubMed2006

Palmitoylation and polymerization of hepatitis C virus NS4B protein.

Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

Cysteine↗
PubMed2006

The effect of sensitisation to insulin with pioglitazone on fasting and postprandial lipid metabolism, lipoprotein modification by lipases, and lipid transfer activities in type 2 diabetic patients.

AIMS/HYPOTHESIS: Insulin resistance is thought to be central to the pathogenesis of diabetic dyslipidaemia. We hypothesised that improving insulin sensitivity would improve fasting and postprandial triglyceride metabolism in patients with type 2 diabetes. To this aim we studied fasting and postprandial lipaemia in type 2 diabetic patients before and after sensitisation to insulin with pioglitazone, compared with that observed in patients on an insulin-providing regime. METHODS: In a double-blind placebo-controlled protocol, 22 patients with type 2 diabetes were randomly allocated to receive either pioglitazone (45 mg/day) or glibenclamide (5 mg/day), for a 20-week period. Fasting and postprandial lipid metabolism were investigated at baseline and at the end of the treatment period. A group of non-diabetic subjects was also studied. RESULTS: Compared with glibenclamide treatment, pioglitazone treatment decreased fasting triglyceride, glucose and insulin levels and the homeostasis model assessment score of insulin resistance. Decreased fasting triglyceride after pioglitazone treatment was due to reduced VLDL triglyceride, particularly VLDL-2. Lipoprotein lipase activity was unchanged by pioglitazone treatment but hepatic lipase showed a significant decrease. Pioglitazone treatment lowered total postprandial triglyceride, as well as chylomicron- and chylomicron-remnant retinyl palmitate levels to normal. Glucose disposal improved but remained abnormal. CONCLUSIONS/INTERPRETATION: Insulin sensitisation with pioglitazone has major effects in restoring postprandial lipaemia to normal, while also correcting fasting hypertriglyceridaemia; both factors may have consequences for atherogenic risk in diabetes.

Blood Glucose↗
PubMed1986

Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions.

The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as --P--OH,--OH, and--NH3+ and hydrogen bond accepting groups such as --P--O- and --COO-. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.

Animals↗
PubMed1999

Purification and characterization of a recombinant Haemophilus influenzae outer membrane phosphomonoesterase e (P4).

Haemophilus influenzae is a common inhabitant of the upper respiratory tract and can cause serious infections of mucosal surfaces. Results from recent studies indicate that this pathogen possesses copious amounts of surface-localized phosphomonoesterase activity mediated by the bacterial lipoprotein e (P4). While the enzyme has previously been purified to apparent homogeneity, purification of large amounts of protein has been prevented by presence of N-terminal lipid modification. Recombinant DNA technology was employed to simultaneously replace the N-terminal lipid modification signal sequence with one for protein secretion without such modification and to place expression of the protein under the control of the T7-inducible promoter. Results from this work show that high levels of phosphomonoesterase activity were achieved after IPTG induction and purified to apparent homogeneity after two chromatography steps. Consistent with loss of the N-terminal lipid modification, the recombinant enzyme was easily extracted from the bacterial membrane and partitioned within the matrix of gel filtration chromatography resin while retaining a denatured molecular weight similar to that of wild-type e (P4). Results from physicochemical characterization suggest that the recombinant protein was similar to wild-type protein in SDS-PAGE-derived molecular weight, primary structure, substrate specificity, pH optimum, and sensitivity or resistance to various inhibitors. Acquisition of sufficient amounts of recombinant P4 was a prelude for studies to elucidate the structure and function of this unusual phosphomonoesterase.

Bacterial Outer Membrane Proteins↗
PubMed1995

Doubly-lipid-modified protein sequence motifs exhibit long-lived anchorage to lipid bilayer membranes.

To understand better the potential functional importance of the dual-lipid modifications found in a number of intracellular proteins of eukaryotes, we have examined how "tenaciously" various doubly-lipid-modified peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membranes. Fluorescent-labeled peptides bearing dual-lipid modifications were incorporated into large unilamellar egg phosphatidylcholine/phosphatidylglycerol vesicles, and the kinetics of spontaneous intervesicle transfer of the lipopeptides were monitored by a fluorescence-dequenching assay. Lipopeptides incorporating the stable "dual-anchor" motif -C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homologous proteins exhibit very slow rates of interbilayer transfer (t1/2 > 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- and -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related intracellular tyrosine kinases and G-protein alpha-subunits and in p21H-ras, respectively. Lipopeptides terminating in an unmethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t1/2 = ca. 10 h). Extrapolating from these results, we estimate that the rate of spontaneous desorption of the corresponding doubly-anchored proteins from membranes should be much slower than that of regulated, protein-mediated release (effected by binding to an "escort" protein or by de-S-acylation). As a result the intracellular distributions of these species (and particularly their targeting to specific intracellular membranes) are likely to be governed (and regulated) primarily by kinetic rather than thermodynamic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1998

PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance.

Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP-PhoQ and PmrA-PmrB. Polymyxin resistance is encoded by the PmrA-PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4' phosphate of lipid A. Two PmrA-PmrB-regulated S. typhimurium loci (pmrE and pmrF) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd) that is predicted to encode a UDP-glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA-PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.

Anti-Bacterial Agents↗
PubMed2001

Post-translational GPI lipid anchor modification of proteins in kingdoms of life: analysis of protein sequence data from complete genomes.

To investigate the occurrence of glycosylphosphatidylinositol (GPI) lipid anchor modification in various taxonomic ranges, potential substrate proteins have been searched for in completely sequenced genomes. We applied the big-pi predictor for the recognition of propeptide cleavage and anchor attachment sites with a new, generalized analytical form of the extreme-value distribution for evaluating false-positive prediction rates. (i) We find that GPI modification is present among lower and higher Eukaryota (approximately 0.5% of all proteins) but it seems absent in all eubacterial and three archaeobacterial species studied. Four other archaean genomes appear to encode such a fraction of substrate proteins (in the range of eukaryots) that they cannot be explained as false-positive predictions. This result supports the possible existence of GPI anchor modification in an archaean subgroup. (ii) The frequency of GPI-modified proteins on various chromosomes of a given eukaryotic species is different. (iii) Lists of potentially GPI-modified proteins in complete genomes with their predicted cleavage sites are available at http://mendel.imp.univie.ac.at/gpi/gpi_genomes.html. (iv) Orthologues of known transamidase subunits have been found only for EUKARYA: Inconsistencies in domain structure among homologues some of which may indicate sequencing errors are described. We present a refined model of the transamidase complex.

Archaea↗
PubMed1999

Increased carbonyl modification by lipids and carbohydrates in diabetic nephropathy.

BACKGROUND: In diabetic nephropathy (DN), possible mediators of untoward effects of hyperglycemia include the advanced glycation end products (AGEs). Indeed, an AGE, carboxymethyllysine (CML), accumulates in expanded mesangial matrix and nodular lesions. An advanced lipoxidation end product (ALE), malondialdehyde-lysine (MDA-lysine), generated on proteins during lipid peroxidation also accumulates in these lesions. As both ALEs and AGEs are formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of lipids and carbohydrates, their colocalization suggests an increased carbonyl modification of proteins. METHODS: To address this hypothesis, human diabetic renal tissues were examined to characterize carbonyl modification of proteins by lipids and carbohydrates: (a) ALEs, MDA-lysine and 4-hydroxynonenal (HNE) protein adduct, derived from lipids, and (b) AGEs, pentosidine and CML, derived from carbohydrates. Furthermore, to elucidate the biological effect of carbonyl modification on primary cultured human and rat mesangial cells, the intracellular protein phosphorylation was examined in the presence of various kinds of carbonyl compounds. RESULTS: The ALE and AGE adducts examined were identified in expanded mesangial matrix and nodular lesions. The exposure of cultured mesangial cells to carbonyl compounds resulted in phosphorylation of tyrosine residues of a number of intracellular proteins. CONCLUSIONS: These data suggest a broad derangement in nonenzymatic biochemistry involving both lipids and carbohydrates exists in diabetic glomerular lesions ("carbonyl stress").

Animals↗
PubMed2006

[Efficacy and safety of extended-release niacin alone or with atorvastatin for lipid profile modification].

OBJECTIVE: To evaluate the efficacy and safety of extended-release niacin (niacin ER) either alone or in combination with atorvastatin for the lipid profile modification in the patients with coronary heart disease (CHD) and its equivalents. METHODS: One hundred and ten patients with CHD and its equivalents with serum total cholesterol (TC) > or = 3.5 mmol/L were randomly assigned into three treatment groups: (1) atorvastatin group (n = 38), receiving atorvastatin 10 mg/d for 8 weeks; (2) niacin ER group (n = 38), given niacin ER 500 mg/d for 4 weeks and then 1000 mg/d for 4 weeks; (3) combination treatment group (n = 34), treated with atorvastatin (10 mg/d) plus niacin ER, with the dose initiating from 500 mg/d, and increasing to 1000 mg/d after 4 weeks, for 8 weeks. The serums lipid profiles and adverse effects were assessed in all the patients before treatment, and 4 and 8 weeks after treatment. RESULTS: (1) After 8 weeks of treatment, the serum level of triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) were reduced by 30% and 16% respectively in the niacin ER group compared with the baseline values (both P < 0.05). After 8 weeks, the TC, low-density lipoprotein cholesterol (LDL-C), and TG in the atorvastatin group decreased by 19%, 26%, and 17% respectively compared with the baseline values (all P < 0.05). Combination treatment decreased the TC, LDL-C, and TG levels by 28%, 38%, and 39% respectively, and increased the HDL-C level by 23% (all P < 0.05). The improvement in TC and LDL-C achieved by combination treatment was superior to treatment of atorvastatin alone and treatment of niacin ER alone (all P < 0.05). (2) The rate of achieving the LDL-C goal of The National Cholesterol Education Program (NCEP) in Adult Treatment Panel III (ATP III) in the combination therapy group was 73.5%, significantly higher than those of the atorvastatin and niacin groups (47.7% and 42.1% respectively, both P < 0.05). (3) Adverse effect, such as flushing (15.8%) and gastrointestinal symptoms (23.7%) were found in the niacin ER group, however, no more adverse effects were found in the combination therapy group. There were no serious adverse events in all groups. CONCLUSION: Niacin ER has a favorable effect in modulating the blood lipid profile, especially in reducing TG and elevating HDL-C. Combined statin with niacin may produce a more global and effective improvement in lipid blood levels than monotherapy and is generally safe and well tolerable.

Aged↗
PubMed1999

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.

Amino Sugars↗
PubMed1994

Effects of ebselen and probucol on oxidative modifications of lipid and protein of low density lipoprotein induced by free radicals.

The oxidative modification of low density lipoprotein (LDL) is accepted to be an important early event of atherosclerosis, but it has not yet been well understood. The preventive effects of two antioxidants with different functions, ebselen and probucol, against the oxidative modification of LDL induced by copper or a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied in order to elucidate the mechanism of modification of apolipoprotein B-100 (apoB). Ebselen inhibited the copper-induced oxidation completely by reducing the hydroperoxides in LDL, since the initiation of copper-dependent oxidation requires the presence of a trace amount of hydroperoxides in LDL. On the other hand, ebselen did not suppress the oxidations of LDL induced by AAPH which generated free radicals by its thermal decomposition. The AAPH-induced oxidation of LDL in the absence of ebselen gave phosphatidylcholine hydroperoxide and cholesteryl ester hydroperoxide as major products, while in its presence, the hydroperoxides were reduced to corresponding alcohols. Interestingly, ebselen had little effect on the increase of relative electrophoretic mobility and fragmentation of intact apoB in the AAPH-induced oxidation. Probucol inhibited the oxidation of lipids in LDL effectively induced by either copper or AAPH, but the protein modifications were observed even in the presence of probucol. It was suggested that (1) lipid hydroperoxides do not play an important role in the modification of apoB such as increase in negative charge and fragmentation, (2) the direct attack of free radicals upon apoB and its modification by lipid oxidation products derived from hydroperoxides increase the negative charge of apoB, and (3) its fragmentation is caused primarily by an attack of free radicals.

Amidines↗
PubMed2004

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

Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance. ArnA is a bifunctional enzyme. It can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. We show that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA. This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme. The crystal structure of the ArnA decarboxylase domain from E. coli is presented here. The structure confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family. On the basis of sequence and structure comparisons of the ArnA decarboxylase domain with other members of the short-chain dehydrogenase/reductase (SDR) family, we propose a binding model for NAD(+) and UDP-glucuronic acid and the involvement of residues T(432), Y(463), K(467), R(619), and S(433) in the mechanism of NAD(+)-dependent oxidation of the 4''-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.

Amino Acid Sequence↗
PubMed2003

Reaction of N(alpha)-hippuryllysine with 2-hydroxyheptanal: a model for lysine-directed protein modifications by lipid peroxidation.

2-Hydroxyheptanal (2-HH) is one of the major aldehydes derived from peroxidation of polyunsaturated fatty acids. In the present study, to obtain an insight into the contributions of 2-HH to protein modifications during lipid peroxidation, a lysine-containing dipeptide, N(alpha)-hippuryllysine (N-benzoylglycyl-L-lysine, BGL), was reacted with 2-HH at neutral pH. The products were characterized on the basis of LC/MS and NMR spectroscopy. The reaction afforded a 2:1 2-HH-lysine adduct, 1-[5-(N-benzoylglycylamino)-5-carboxypentyl]-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one (I). In addition, we obtained a 1:1 2-HH-lysine adduct, N-[5-(N-benzoylglycylamino)-5-carboxypentyl]-1-amino-2-heptanone (III). The treatment of the purified III with 2-HH produced I. On the other hand, when the reaction mixture was allowed prolonged standing, I was slowly oxidized to 1-[5-(N-benzoylglycylamino)-5-carboxypentyl]-4-butyl-5-pentyl-3-hydroxypyridinium (V). This conversion was strongly accelerated by the addition of copper(II) ion and 2,2'-bipyridyl. We propose here that the above series of conversions is the main pathway for the modification of lysine residues of proteins by 2-HH.

Aldehydes↗
PubMed2001

[Correlation between severity of angina, its stability, and oxidative modification of lipids in patients with ischemic heart disease].

AIM: To elucidate the relations between angina severity, stability, concomitant arterial hypertension, diabetes mellitus, plasma oxidation. MATERIAL AND METHODS: The study included 152 patients with stable angina of effort (functional class II-III). Of them, 67 had concomitant arterial hypertension (AH), 20 AH and compensated diabetes mellitus type II, 14 patients with anginal functional class III were admitted to hospital for unstable angina, 11 patients with IHD with unstable angina and 28 healthy donors. The study was made of dynamic features of lipid oxidation using copper-induced plasma oxidation. Lipids were measured with enzyme assays. RESULTS: The degree of LP oxidation increases with aggravation of angina. IHD risk factors activate atherogenic modification of lipids in patients with stable angina. There was no differences between the groups in content of low density lipoproteins cholesterol. CONCLUSION: Plasma resistance to oxidation may serve an additional criterion for assessment of severity and stability of various IHD forms.

Angina Pectoris↗
PubMed1996

A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein - partial purification and characterization from rat brain.

The Rho subfamily, consisting of three members (RhoA, -B and -C), belongs to the small GTP-binding protein superfamily. The Rho subfamily is implicated in regulation of various actin filament-dependent cell functions, such as cell aggregation, cell motility and cytokinesis. The Rho subfamily receives an upstream signal and is converted from the GDP-bound inactive form to the GTP-bound active form which transduces a signal to a downstream pathway. This conversion is regulated by GDP/GTP exchange proteins (GEPs) and several GEPs for the Rho subfamily have been identified. The GEPs thus far reported are mainly isolated from the cytosol fraction of various tissues and are not specific for the Rho subfamily. Here we have partially purified a membrane-associated GEP specific for the Rho subfamily (mRho GEP). mRho GEP was extracted from the crude synaptic membrane fraction of rat brain by a combination of detergent and NaCl, and partially purified by several column chromatographies. The partially purified mRho GEP was active on RhoA but was inactive on other small GTP-binding proteins including at least Rac1, Ki-Ras and Rab3A. RhoA undergoes post-translational lipid modifications and mRho GEP required these lipid modifications for its GEP activity. mRho GEP was not active in the presence of Rho GDI, an inhibitory Rho GEP. These results indicate that there is a membrane-associated GEP specific for Rho and suggest that Rho is activated by this GEP on the membranes.

Animals↗
PubMed2005

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 approximately 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 1H and 13C 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.

Amino Sugars↗