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Research Interests: Biochemistry, Chemistry, Biology, Biological Chemistry, Medicine, and 12 moreBiological Sciences, Mutation, Escherichia coli, Temperature, CHEMICAL SCIENCES, Protein Conformation, Glycine, Amino Acid Sequence, Biotinylation, Acetyl CoA Carboxylase, Molecular Sequence Data, and Medical and Health Sciences
Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated... more
Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutar...
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10.1074/jbc.M110.194191 Access the most updated version of this article at doi: . JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the Alerts: When a correction for this article is posted • When... more
10.1074/jbc.M110.194191 Access the most updated version of this article at doi: . JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the Alerts: When a correction for this article is posted • When this article is cited • to choose from all of JBC's e-mail alerts Click here http://www.jbc.org/content/286/10/8263.full.html#ref-list-1 This article cites 45 references, 15 of which can be accessed free at
Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter... more
Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofa...
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Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N -(3-oxohexanoyl)- l... more
Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N -(3-oxohexanoyl)- l -homoserine lactone from an acyl-acyl carrier protein and S -adenosylmethionine. Another V. fischeri gene, ainS , directs the synthesis of N -octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N -(3-hydroxybutyryl)- l -homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N -octanoylhomoserine lactone from S -adenosylmethionine and either octanoyl-acyl carrier protei...
Research Interests: Biochemistry, Bacteriology, Kinetics, Biology, Medicine, and 15 moreBiological Sciences, Quorum Sensing, ATP synthase, Enzyme, Vibrio, Substrate Specificity, S-Adenosylmethionine, Vibrio Fischeri, Reaction Kinetics, Fusion Protein, Lactones, Acyl carrier protein, Sequence similarity, Medical and Health Sciences, and Maltose binding protein
BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in... more
BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l -alanine in a pyridoxal 5′-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l -alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilize...
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Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase)... more
Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were dete...
Research Interests: Biochemistry, Kinetics, Biology, Medicine, Biological Sciences, and 15 moreEscherichia coli, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Gel Permeation Chromatography, CHEMICAL SCIENCES, Substrate Specificity, Cysteine, DNA ligase, Amino Acid Sequence, Cations, Hydrolysis, Hydrogen-Ion Concentration, Molecular Sequence Data, gene amplification, and Medical and Health Sciences
BioH is an α/β-hydrolase required for synthesis of the pimelate moiety of biotin in diverse bacteria. The bioH gene is found in different genomic contexts. In some cases (e.g., Escherichia coli) the gene is not located within a biotin... more
BioH is an α/β-hydrolase required for synthesis of the pimelate moiety of biotin in diverse bacteria. The bioH gene is found in different genomic contexts. In some cases (e.g., Escherichia coli) the gene is not located within a biotin synthetic operon and its transcription is not coregulated with the other biotin synthesis genes. In other genomes such as Pseudomonas aeruginosa the bioH gene is within a biotin synthesis operon and its transcription is coregulated with the other biotin operon genes. The esterases of pimelate moiety synthesis show remarkable genomic plasticity in that in some biotin operons bioH is replaced by other α/ß hydrolases of diverse sequence. The "wild card" nature of these enzymes led us to compare the paradigm "freestanding" E. coli BioH with the operon-encoded P. aeruginosa BioH. We hypothesized that the operon-encoded BioH might differ in its expression level and/or activity from the freestanding BioH gene. We report this is not the cas...
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Helicobacter pylori is a Gram-negative bacterium that inhabits the upper gastrointestinal tract in humans, and the presence of this pathogen in the gut microbiome increases the risk of peptic ulcers and stomach cancer. H. pylori depends... more
Helicobacter pylori is a Gram-negative bacterium that inhabits the upper gastrointestinal tract in humans, and the presence of this pathogen in the gut microbiome increases the risk of peptic ulcers and stomach cancer. H. pylori depends on unsaturated fatty acid (UFA) biosynthesis for maintaining membrane structure and function. Although some of the H. pylori enzymes involved in UFA biosynthesis are functionally homologous with the enzymes found in Escherichia coli, we show here that an enzyme HP0773, now annotated as FabX, uses an unprecedented backtracking mechanism to not only dehydrogenate decanoyl-acyl carrier protein (ACP) in a reaction that parallels that of acyl-CoA dehydrogenase, the first enzyme of the fatty acid β-oxidation cycle, but also isomerizes trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, the key UFA synthetic intermediate. Thus, FabX reverses the normal fatty acid synthesis cycle in H. pylori at the C10 stage. Overall, this unusual FabX activity may offer a broader ...
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In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the... more
In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was d...
Research Interests: Biochemistry, Biology, Helicobacter pylori, Medicine, Escherichia coli, and 4 moreGene, Biotin, Enzyme, and Biosynthesis
Research Interests: Molecular Evolution, Biology, Biological Chemistry, Medicine, Biological Sciences, and 12 morePhylogeny, Sequence alignment, Escherichia coli, Streptomyces coelicolor, Enzyme, CHEMICAL SCIENCES, DNA ligase, Amino Acid Sequence, Adenosine monophosphate, Molecular Sequence Data, thioctic acid, and Medical and Health Sciences
Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on... more
Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation ...
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Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both... more
Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium. Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties. However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml. This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate. Labeling experiments with [75Se]selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells. Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated. Two of these isol...
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Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an... more
Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis , expression of one gene from this pathway, lipB , encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic re...
Research Interests: Biochemistry, Chemistry, Mass Spectrometry, Medicine, Multidisciplinary, and 13 moreCrystal structure, Tuberculosis, Mycobacterium tuberculosis, Enzyme, Protein Complex Detection, Active site, Lysine, Alpha Lipoic Acid, Cysteine, Atomic Resolution, Acyltransferase, Acyl carrier protein, and Multiple drug-resistant
Research Interests: Biochemistry, Chemistry, Plant Biology, Biology, Plant cell physiology, and 15 moreMembrane Proteins, Medicine, Gene expression, Arabidopsis thaliana, Mutagenesis, Escherichia coli, Arabidopsis, Molecular cloning, Lipoproteins, Plant cell, Amino Acid Sequence, Base Sequence, Biochemistry and cell biology, Molecular Sequence Data, and thioctic acid
Research Interests: Protein Folding, Kinetics, Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Molecular Recognition, Escherichia coli, Biological, Plasmids, CHEMICAL SCIENCES, Amino Acid Sequence, Amino Acid Substitution Rates, Structure activity Relationship, Protein Binding, Biotinylation, Acetyl CoA Carboxylase, Molecular Sequence Data, binding sites, and Medical and Health Sciences
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, and 8 moreCHEMICAL SCIENCES, Molecular cloning, Substrate Specificity, Enterococcus faecalis, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
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Research Interests: Biochemistry, Biology, Biological Chemistry, Medicine, Biological Sciences, and 13 moreEscherichia coli, Temperature, Plasmids, Biotin, CHEMICAL SCIENCES, Amino Acid Sequence, Protein Binding, Site-directed Mutagenesis, Biotinylation, Linker, Acetyl CoA Carboxylase, Molecular Sequence Data, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Biology, Biological Chemistry, Medicine, and 12 moreBiological Sciences, Mutation, Escherichia coli, Temperature, CHEMICAL SCIENCES, Protein Conformation, Glycine, Amino Acid Sequence, Biotinylation, Acetyl CoA Carboxylase, Molecular Sequence Data, and Medical and Health Sciences
Based on its genome sequence, the pathway of β-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that... more
Based on its genome sequence, the pathway of β-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR ) of both organisms in which the genes of fatty acid degradation ( fad ) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to gr...
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Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated... more
Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.
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Plant and fungal mitochondria contain type II fatty acid synthesis systems closely related to those of bacteria in which the individual reactions are catalyzed by separate soluble proteins acting on intermediates bound to acyl carrier... more
Plant and fungal mitochondria contain type II fatty acid synthesis systems closely related to those of bacteria in which the individual reactions are catalyzed by separate soluble proteins acting on intermediates bound to acyl carrier protein (ACP). Mammalian mitochondria are thought to synthesize fatty acids, but evidence for the key ACP component was lacking since the only reported ACP was the SDAP subunit of the membrane‐bound NADH:ubiquinone oxidoreductase, We report that most of the SDAP is found in the soluble (matrix) fraction of bovine heart mitochondria and is therefore available to carry the intermediates of type II fatty acid synthesis.
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Research Interests: Biochemistry, Chemistry, Biology, Medicine, Escherichia coli, and 13 moreIron, Sulfur, Molecular cloning, Biosynthesis, Acylation, American Chemical Society, S-Adenosylmethionine, Oxidation-Reduction, Dithionite, Biochemistry and cell biology, Acyl carrier protein, thioctic acid, and Medical biochemistry and metabolomics
Ralstonia solanacearum , a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C 16:0 ),... more
Ralstonia solanacearum , a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C 16:0 ), palmitoleic (C 16:1 ) and cis -vaccenic (C 18:1 ) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes ( fabF1 , fabF2 , fabF3 , and fabF4 ) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene ( fabB ) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1 , encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynth...
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Research Interests: Biochemistry, Chemistry, Biology, Medicine, Gene, and 3 moreEnzyme, Alpha Lipoic Acid, and Nonsense Mutation
Research Interests: Mass Spectrometry, Biology, Biological Chemistry, Medicine, Sequence Analysis, and 11 moreBiological Sciences, Escherichia coli, Gene, CHEMICAL SCIENCES, Molecular cloning, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Molecular Sequence Data, thioctic acid, and Medical and Health Sciences
Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate. Extracts of two of the mutants... more
Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate. Extracts of two of the mutants were shown to contain normal levels of pyruvate oxidase antigen, although the enzymatic activities of the extracts were reduced or absent. The poxB locus was mapped by using closely linked transposon insertions to min 18.7 of the E. coli linkage map between the cmlA and aroA loci, a location far removed from that of the regulatory gene, poxA.
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Research Interests: Biochemistry, Biology, Phospholipids, Medicine, Enzymes, and 15 moreFatty Acid Synthesis, Fatty acids, Escherichia coli, Membrane Lipids, Biotin, Anti-Bacterial Agents, Biosynthesis, Fatty Acid, Microbiological, Amino Acid Sequence, Lipid A, Molecular Sequence Data, Acyl carrier protein, thioctic acid, and polyketide
AcpB homologs are encoded by many, but not all, lactic acid bacteria ( Lactobacillales ), including many members of the human microbiome. The mechanisms regulating fatty acid synthesis by exogenous fatty acids play a key role in... more
AcpB homologs are encoded by many, but not all, lactic acid bacteria ( Lactobacillales ), including many members of the human microbiome. The mechanisms regulating fatty acid synthesis by exogenous fatty acids play a key role in resistance of these bacteria to those antimicrobials targeted at fatty acid synthesis enzymes. Defective regulation can increase resistance to such inhibitors and also reduce pathogenesis.
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Many Bacteria and Archaea employ the heterodisulfide reductase (Hdr)-like sulfur oxidation pathway. The relevant genes are inevitably associated with genes encoding lipoate-binding proteins (LbpA). Here, deletion of the gene identified... more
Many Bacteria and Archaea employ the heterodisulfide reductase (Hdr)-like sulfur oxidation pathway. The relevant genes are inevitably associated with genes encoding lipoate-binding proteins (LbpA). Here, deletion of the gene identified LbpA as an essential component of the Hdr-like sulfur-oxidizing system in the Alphaproteobacterium Hyphomicrobium denitrificans. Thus, a biological function was established for the universally conserved cofactor lipoate that is markedly different from its canonical roles in central metabolism. LbpAs likely function as sulfur-binding entities presenting substrate to different catalytic sites of the Hdr-like complex, similar to the substrate-channeling function of lipoate in carbon-metabolizing multienzyme complexes, for example pyruvate dehydrogenase. LbpAs serve a specific function in sulfur oxidation, cannot functionally replace the related GcvH protein in Bacillus subtilis and are not modified by the canonical E. coli and B. subtilis lipoyl attachme...
Research Interests: Biochemistry, Chemistry, Biology, Medicine, Archaea, and 4 moreBacteria, Sulfur, Bacillus subtilis, and Elife
The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing... more
The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, , , and Although physiological roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiologically relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degradation. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochemically defined.
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The precursors of the diffusible signal factor (DSF) family signals of pv. are 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway.... more
The precursors of the diffusible signal factor (DSF) family signals of pv. are 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by the pv. XCC0416 gene (), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of an temperature-sensitive strain. However, when transformed into the strain together with a plasmid bearing the acyl-ACP synthetase gene (), growth proceeded, but only when the medium contained octanoic acid. assays showed that FabG2 catalyzes the reduction of long-chain (≥C) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C, C) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a s...
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Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur... more
Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). In Bacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. The Escherichia coli pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising that E. coli GcvH functional...
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SUMMARY Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become... more
SUMMARY Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli , which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis , which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage s...
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Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metabolism were characterized by DNA sequence analysis. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a... more
Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metabolism were characterized by DNA sequence analysis. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 25-kDa protein. Overproduction of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that Lip...
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This review presents the most thoroughly studied bacterial fatty acid synthetic pathway, that of Escherichia coli and then discusses the exceptions to the E. coli pathway present in other bacteria. The known interrelationships between the... more
This review presents the most thoroughly studied bacterial fatty acid synthetic pathway, that of Escherichia coli and then discusses the exceptions to the E. coli pathway present in other bacteria. The known interrelationships between the fatty acid and polyketide synthetic pathways are also assessed, mainly in the Streptomyces group of bacteria. Finally, we present a compendium of methods for analysis of bacterial fatty acid synthetic pathways.
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Research Interests: Biochemistry, Microbiology, Biology, Phospholipids, Biological Chemistry, and 13 moreMedicine, Biological Sciences, DNA, Fatty acids, Mutation, Escherichia coli, Plasmids, CHEMICAL SCIENCES, Multienzyme complexes, Enterococcus faecalis, Cell free System, Acyl carrier protein, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Organelles, Fatty acids, Animals, Lipid metabolism, CHEMICAL SCIENCES, In Vivo, Biosynthesis, Plasmodium falciparum, Enterococcus faecalis, Protozoan Proteins, Lactococcus lactis, Apoproteins, Acyl carrier protein, and Medical and Health Sciences
One of the mutants ( slr7 mutant) of a wild-type Escherichia coli strain resistant to selenolipoic acid reported previously (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) unexpectedly grew... more
One of the mutants ( slr7 mutant) of a wild-type Escherichia coli strain resistant to selenolipoic acid reported previously (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) unexpectedly grew on minimal medium following transductional introduction of a lipA null mutation. We report that the slr7 strain carries a duplication of the lip chromosomal region that causes the phenotype of the mutant strain.
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The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli , transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid... more
The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli , transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene ( lplA ) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced K m values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.