Biosynthesis of iso-fatty acids in myxobacteria: iso-even fatty acids are derived by alpha-oxidation from iso-odd fatty acids

The biosynthesis of iso-fatty acids was investigated in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca by feeding labeled precursors of these fatty acids and by conducting detailed analysis of the fatty acid profiles and fatty acid MS and NMR spectra. These experiments revealed that in S. aurantiaca all iso-even fatty acids are formed via alpha-oxidation of iso-odd fatty acids and not by using valine-derived isobutyryl-CoA.     

A novel type of geosmin biosynthesis in myxobacteria

The biosynthesis of geosmin (1) and (1(10)E,5E)-germacradien-11-ol (2), two volatile terpenoid compounds emitted by the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca, was investigated in feeding experiments with different labeled precursors. In these experiments, the volatiles released by the cell cultures grown on agar plates were collected with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. [(2)H(10)]Leucine and [4,4,4,5,5,5-(2)H(6)]dimethylacrylate were fed to wild-type strains and bkd mutant strains, which are impaired in the degradation of leucine to isovaleryl-CoA. [(2)H(10)]Leucine was incorporated into 1 and 2 only by the wild-type strains via the biosynthetic pathway that involves leucine degradation and branching into the mevalonate pathway. Dimethylacrylyl-CoA (DMA-CoA) is an intermediate in the leucine degradation and in the recently discovered pathway from HMG-CoA to isovaleryl-CoA. The corresponding free acid, [4,4,4,5,5,5-(2)H(6)]dimethylacrylic acid, was incorporated into 1 and 2 only by the mutants impaired in leucine degradation. [4,4,6,6,6-(2)H(5)]Mevalonic acid lactone (12) was synthesized and fed to M. xanthus and S. aurantiaca wild-type strains and a double mutant strain of M. xanthus. This strain does not degrade leucine and is impaired in the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid. The mass spectral analysis of labeled 1 and 2 obtained in these feeding experiments led to a biosynthetic scheme to 1 with intermediate 2. This pathway differs from that observed in the liverwort Fossombronia pusilla and thus suggests microbial geosmin biosynthesis following a route different from that in liverworts. Our results are supported by a 1,2-hydride shift of the tertiary hydrogen atom at C-4a into the ring opposite to that in F. pusilla.     

Racemic D,L-asparagine causes enantiomeric excess of other coexisting racemic D,L-amino acids during recrystallization: a hypothesis accounting for the origin of L-amino acids in the biosphere

When recrystallizations were performed using a mixture of 12 D,L-amino acids (alanine, aspartic acid, arginine, glutamic acid, glutamine, histidine, leucine, methionine, serine, valine, phenylalanine, and tyrosine) with excess D,L-asparagine, all amino acids with the same configuration as asparagine were preferentially co-crystallized, indicating that it is the nature of a mixture of racemic amino acids to produce a spontaneous high enantiomeric excess.     

Production of branched-chain alkylprodiginines in S. coelicolor by replacement of the 3-ketoacyl ACP synthase III initiation enzyme, RedP

The enzyme RedP is thought to initiate the biosynthesis of the undecylpyrolle component of the antibiotic undecylprodiginine produced by Streptomyces coelicolor. RedP has homology to FabH, which initiates fatty acid biosynthesis by condensing the appropriate acyl-CoA starter unit with malonyl ACP. We have generated a redP-deletion mutant of S. coelicolor M511 (SJM1) and shown that it produces reduced levels of prodiginines and two new analogs, methylundecylprodiginine and methyldodecylprodiginine. Incorporation studies with perdeuterated valine were consistent with these being generated using methylbutyryl-CoA and isobutyryl-CoA as starter units, respectively. Plasmid-based expression of a streptomycete fabH in the SJM1 mutant led to restoration of overall prodiginine titers but the same overall ratio of undecylprodiginines and novel prodiginines. Thus, the redP FabH can be replaced by FabH enzymes with different substrate specificities and provides a method for generating novel prodiginines.     

Microstructural characterization of terpolymers of leucine, β‐benzyl aspartate,and valine

Three different characterization methods—¹³C NMR spectroscopy, a terminal terpolymerization model, and a probability analysis based on the Poisson distribution—were used to determine the microstructure of random terpolymers. The methods were used to determine the amino acid sequence distribution of random terpolymers prepared from the polymerization of N‐carboxyanhydrides that contained L ‐leucine, β‐benzyl‐L ‐aspartate, and L ‐valine. Poly(L ‐leucine‐L ‐aspartic acid‐L ‐valine) [poly(LDV)] was designed as a target specific substrate for the α₄β₁ integrin that recognizes the tripeptide sequence leucine‐aspartic acid‐valine (LDV). The presence of the tripeptide sequence LDV within the polymer was determined to be eight LDV triad sequences on average in terpolymers of approximately 100 kDa. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4328–4337, 2006     

Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids

2-Hexadecynoic acid and 2-octadecynoic acid have cidal activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. At subinhibitory concentrations, M. smegmatis rapidly transformed [1-(14)C]-2-hexadecynoic acid into endogenous fatty acids and elongated them into mycolic acids. Toxic concentrations of 2-hexadecynoic acid resulted in accumulation of 3-ketohexadecanoic acid, which blocked fatty acid biosynthesis, and 3-hexadecynoic acid, an inhibitor of fatty acid degradation. The combination of these two metabolites is necessary to achieve the inhibition of M. smegmatis. We conclude that 2- and 3-hexa/octadecynoic acids inhibit mycolic acid biosynthesis, fatty acid biosynthesis, and fatty acid degradation, pathways of significant importance for mycobacteria.     

Valine and phenylalanine as precursors in the biosynthesis of alkamides in Acmella radicans

Acmella radicans (Asteraceae) produces at least seven alkamides, most with either an isobutyl- or phenylethyl group as the amine moiety. These moieties suggest that the amino acids valine and phenylalanine are the biosynthetic precursors of these alkamides. On the basis of labeled feeding experiments using either L-[2H8]valine or L-[2H8]phenylalanine we present evidence for the involvement of these two amino acids in the biosynthesis of (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (affinin) (1), (2Z,4E)-N-(2-phenylethyl)-2,4-octadienamide (2), (2E)-N-(2-phenylethyl)-nona-2-en-6,8-diynamide (3), and 3-phenyl-N-(2-phenylethyl)-2-propenamide (4). Alkamides were isolated from young A. radicans plants and analyzed by gas chromatography-mass spectrometry (GC-MS). Additionally, in cell free in vitro experiments based on isobutyl and phenylethylamide biosynthesis, using a colorimetric assay and GC-MS, valine and phenylalanine decarboxylase activities were assayed in the soluble extract of A. radicans leaves.     

Water‐Mediated Recognition of Simple Alkyl Chains by Heart‐Type Fatty‐Acid‐Binding Protein

Long‐chain fatty acids (FAs) with low water solubility require fatty‐acid‐binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub‐Angstrom X‐ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart‐type FABP (FABP3) preferentially incorporates a U‐shaped FA of C10–C18 using a lipid‐compatible water cluster, and excludes longer FAs using a chain‐length‐limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.     

Creation and evaluation of a two-dimensional contour plot of fatty acid methyl esters after off-line coupling of reversed-phase HPLC and GC/EI-MS

Fatty acid methyl esters obtained from a fish oil sample were fractionated by non-aqueous reversed-phase high-performance liquid chromatography (RP-HPLC) using three serially connected C₁₈-columns and pure methanol as the eluent. The HPLC fractions were analyzed by gas chromatography–electron ionization mass spectrometry in the selected ion monitoring mode. Data analysis and visualization was performed by the creation of a two-dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. The 2D contour plot resulted in a full resolution of more than 120 fatty acids. The fatty acids were arranged on predictable lines and curves in dependence of the number of carbons and double bonds. The 2D contour plot enabled both the recognition of unknown fatty acids (which were found off the lines and curves) and the prediction of the coordinates of known fatty acids. Finally, selected HPLC fractions were subjected to further experiments (hydrogenation, silver ion fractionation, specific GC/MS measurements) in order to verify the structural assignments predicted from the 2D contour plot. All in all, the structures of over 100 FAs could be assigned to the peaks detected in the 2D contour plots.     

Final elucidation of the absolute configuration of the signal metabolite hormaomycin

The complete absolute configuration of hormaomycin 1 a has been established by HPLC and HPLC/MS experiments with appropriately derivatized 4-propylprolines, (2S,4S)-6 and (2R,4R)-6, as well as 4-(Z)-propenylprolines, cis-5 and trans-5, and also feeding experiments with enantiomerically pure samples of the deuterium-labeled 3-(2'-nitrocyclopropyl)alanine, (2S)-3,3-[D2]15 and (2S)-2,2'-[D2]15, and 4-(Z)-propenylproline 2',4-[D2]-(2S,4R)-5. The latter five amino acids were prepared for the first time and allowed one to unequivocally assign the hitherto unknown absolute configurations of the last four stereocenters in hormaomycin 1 a. As a bonus, some new information about the biosynthesis of this molecule has also been gathered.     

Biosynthetic precursors of the lipase inhibitor lipstatin

Three putative intermediates in the biosynthesis of the lipase inhibitor lipstatin were synthesized in stable isotope-labeled form and were added to fermentation cultures of Streptomyces toxytricini. Biosynthetic lipstatin was isolated and analyzed by NMR spectroscopy. [3,10,11,12-(2)H]-(3S,5Z,8Z)-3-hydroxytetradeca-5,8-dienoic acid (9) was shown to serve as a direct biosynthetic precursor of lipstatin. [7,8-(2)H(2)]Hexylmalonate (11) was also incorporated into lipstatin, albeit at a relatively low rate. The leucine moiety of [(13)C-formyl,(15)N]-N-formylleucine (10) was diverted to lipstatin under loss of the (13)C-labeled formyl residue.     

31P NMR spectroscopy as a powerful tool for the determination of enantiomeric excess and absolute configurations of alpha-amino acids

An easy method for the determination of the enantiomeric excess (ee) of mixtures of alpha-amino acids, and also for the elucidation of the absolute configuration of each component of the mixture, is reported. The method is based on the formation of diastereoisomers by reaction of the enantiomerically pure acetylacetonate derivative [Pd(acac-O,O')(P(2)-dach)]ClO(4) (4) [P(2)-dach = (1R,2R)-C(6)H(10)(NHPPh(2))(2)] with d,l-mixtures of alpha-amino acids AaH (Pd:AaH = 1:1 molar ratio, refluxing MeOH). The reaction occurs with protonation of the acac ligand and N,O-coordination of the amino acidate group, giving the corresponding [Pd(Aa-N,O)(P(2)-dach)]ClO(4) complexes l-5 and d-6. The composition of these mixtures of amino acidate complexes was analyzed by integration of the corresponding peaks (four doublets, two for each diastereomer) in their (31)P((1)H) NMR spectra. A series of 14 alpha-amino acids was studied (a, alanine; b, 2-aminobutyric acid; c, valine; d, phenylalanine; e, proline; f, leucine; g, isoleucine; h, norleucine; i, serine; j, threonine; k, methionine; l, aspartic acid; m, glutamine; n, cysteine), and excellent agreement between the expected values of ee and those obtained from integration of the (31)P((1)H) NMR spectra was obtained. Moreover, the position of the signals of each isomer is diagnostic, in such a way that the outer doublets are always due to the l-derivatives 5a-l, while the inner ones are due to the d-derivatives 6a-l, allowing the assignation of absolute configurations to each isomer in the mixture.     

Gas-liquid chromatographic method for analysing complex mixtures of fatty acids including conjugated linoleic acids (cis9trans11 and trans10cis12 isomers) and long-chain (n-3 or n-6) polyunsaturated fatty acids. Application to the intramuscular fat of beef meat

The optimisation and validation of a gas-liquid chromatographic (GLC) method using direct saponification with KOH/methanol followed by a derivatization with (trimethylsilyl)diazomethane was carried out trying to overcome all the difficulties posed by the analysis of complex mixtures of fatty acids (FAs) in animal fat tissues. The presented method allowed sensitive, selective and simultaneous determination of a wide range of different FAs, including short-chain FAs, branched-chain FAs and conjugated linoleic acid isomers in the same GLC run along with other well known saturated, monounsaturated and polyunsaturated FAs. To demonstrate the feasibility of the procedure, the total FA profile of beef meat was characterised.     

Acid-base thermochemistry of gaseous aliphatic α-aminoacids

Acid-base thermochemistry of isolated aliphatic amino acids (denoted AAA): glycine, alanine, valine, leucine, isoleucine and proline has been examined theoretically by quantum chemical computations at the G3MP2B3 level. Conformational analysis on neutral, protonated and deprotonated species has been used to identify the lowest energy conformers and to estimate the population of conformers expected to be present at thermal equilibrium at 298 K. Comparison of the G3MP2B3 theoretical proton affinities, PA, and ΔH(acid) with experimental results is shown to be correct if experimental thermochemistry is re-evaluated and adapted to the most recent acidity-basicity scales. From this point of view, a set of evaluated proton affinities of 887, 902, 915, 916, 919 and 941 kJ mol(-1), and a set of evaluated ΔH(acid) of 1433, 1430, 1423, 1423, 1422 and 1426 kJ mol(-1), is proposed for glycine, alanine, valine, leucine, isoleucine and proline, respectively. Correlations with structural parameters (Taft's σ(α) polarizability parameter and molecular size) suggest that polarizability of the side chain is the major origin of the increase in PA and decrease in ΔH(acid) along the homologous series glycine, alanine, valine and leucine/isoleucine. Heats of formation of gaseous species AAA, AAAH(+) and [AAA-H](-) were computed at the G3MP2B3 level. The present study provides previously unavailable Δ(f)H°(298) for the ionized species AAAH(+) and [AAA-H](-). Comparison with Benson's estimate, and correlation with molecular size, show that several experimental Δ(f)H°(298) values of neutral or gaseous AAA might be erroneous.     

Synthesis and evaluation of radioiodinated terminal-substituted 5-iodo-(2-thienyl) fatty acids as new myocardial imaging agents

In order to evaluate the stability of radioiodide attached to the 5 position of thiophenes substituted at the 2-position with tissue-specific groups as new radiopharmaceuticals, two [¹²⁵I]iodothienyl-substituted long-chain fatty acids have been prepared and evaluated in rats. Radioiodide was introduced into the 5 position of 17-(2-thienyi)heptadecanoic acid and 13-(2-thienyl)tridecanoic acid by K-¹²⁵I treatment of their corresponding 5-[bis-(trifluoroacetoxy)]thallium derivatives. Tissue distribution studies in rats with 17-[5-[¹²⁵I]iodo(2-thienyl)]heptadecanoic acid shows significant heart uptake and prolonged retention accompanied by in vivo deiod-ination and moderate blood levels. A comparison of the heart uptake of the 17 carbon fatty acid with a 13 carbon analogue, 13-[5-[¹³⁵I]iodo(2-thienyl)]tridecanoic acid, demonstrated a significantly greater myocardial uptake for the 17 carbon fatty acid than the 13 carbon analogue. These results suggest that the 5-iodothienyl moiety substituted at the terminal position of long chain fatty acids does not interfere with myocardial uptake and that such compounds may be of value as a new class of myocardial imaging agents.     

Palladium-catalyzed alpha-arylation of azlactones to form quaternary amino acid derivatives

[reaction: see text] The synthesis of alpha-aryl-alpha-alkyl amino acid derivatives from alpha-amino acids by the arylation of azlactone derivatives is reported. Arylation of azlactones derived from alanine, valine, phenalanine, phenyl glycine, and leucine all provided good yields of the arylated product. Mechanistic studies of this reaction revealed that a stable complex containing a ligand formed by reaction of dba with the azlactone accounts for a new inhibiting effect of dba when reactions are initiated with Pd(dba)(2).     

Biosynthesis and structural revision of neomarinone

[reaction: see text] The biosynthesis of the meroterpenoid neomarinone from a marine actinomycete was probed through feeding experiments with (13)C-labeled precursors. NMR characterization of [U-(13)C(6)]glucose-enriched neomarinone led to the structural revision of structure 4a to 4b, which was confirmed by extensive 2D NMR spectrometry with unlabeled compound.     

Stereospecificity of an enzymatic monoene 1,4-dehydrogenation reaction: conversion of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid

In this article, we report the first stereochemical study of an enzymatic 1,4-dehydrogenation reaction, namely, the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, involved in the sex pheromone biosynthesis of the moth Spodoptera littoralis. The investigation was carried out using the labeled substrates (R)-[10-(2)H]- and (S)-[10-(2)H]-tridecanoic acids ((R)-2 and (S)-2, respectively) and (R)-[2,2,3,3,13-(2)H(5)]- and (S)-[2,2,3,3,13-(2)H(5)]-tetradecanoic acids ((R)-1 and (S)-1, respectively). Probes (R)-2 and (S)-2 were prepared as described in a previous article.(1) The synthesis of the pentadeuterated chiral substrates (R)-1 and (S)-1 was accomplished by kinetic resolution of the racemic 12-tridecyn-2-ol (6) with immobilized porcine pancreatic lipase. The enantiomerically pure alcohols (R)-6 and (S)-6 were transformed into the final acids (S)-1 and (R)-1, respectively, by a sequence of well-established reactions. The analyses of methanolyzed lipidic extracts from glands incubated separatedly with each individual probe showed that in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, both pro-(R) hydrogen atoms at C-10 and C-13 are removed from the substrate. This is the first example reported of a desaturase with pro-(R)/pro-(R) stereospecificities that gives rise to (E)-double bonds. A mechanistic explanation for the stereochemical outcome of this reaction is advanced.     

气相色谱测定牦牛骨中脂肪酸的甲酯化方法比较

利用气相色谱（GC）技术,采用酸水解提取脂质,比较了6种甲酯化法（乙酰氯-甲醇法、H₂SO₄-甲醇法、HCl-甲醇法、KOH-甲醇法、KOH-甲醇+H₂SO₄-甲醇法和KOH-甲醇+HCl-甲醇法）对脂肪酸测定的影响,优选牦牛骨中脂肪酸测定的最佳方法。37种脂肪酸标准样品在0.28~250.00 mg/L范围内线性关系良好,相关系数均大于0.99（除C4：0外）。碱酯化法和酸碱结合法几乎无法测出牦牛骨中的脂肪酸,其测得的总脂肪酸含量小于0.20 g/100 g。乙酰氯-甲醇法测得的总脂肪酸含量（13.61 g/100 g）显著高于H₂SO₄-甲醇法（总脂肪酸含量为11.68 g/100 g）和HCl-甲醇法（总脂肪酸含量为3.18 g/100 g）测得的结果。乙酰氯-甲醇法和H₂SO₄-甲醇法的日内和日间精密度分别为0.27%~8.60%和0.34%~2.64%,两种方法中脂肪酸的回收率为83.06%~105.54%。结果表明,酸水解-乙酰氯-甲醇法是牦牛骨中脂肪酸测定的最佳方法。C18：1n9c、C16：0、C18：0和共轭亚油酸（CLA）是牦牛骨的主要脂肪酸,其总和达脂肪酸总量的85%以上,饱和脂肪酸与不饱和脂肪酸含量比值约为1：2。牦牛骨中脂肪酸的研究为骨资源脂质的有效利用提供了重要依据。     

Site specificity in the H–D exchange reactions of gas-phase protonated amino acids with CH3OD

H–D exchange reactions of methanol-d₁ with protonated amino acids were performed in an external-source Fourier transform mass spectrometer. Absolute rate constants were determined for the group which included glycine, alanine, valine, leucine, isoleucine and proline. By comparing reactivities with selected methyl esters, it was found that exchange on the carboxylic acid occurs 3–10 times faster than exchange on the amino group. No simple correlation is observed between the rates of H–D exchange on the acid group and the size of the alkyl group. However, the rates of exchange on the amine decrease with increasing gas-phase basicity. Glycine, the least basic amino acid, exchanges its amine hydrogens the fastest. These results are useful for determining the interaction of methanol with protonated amino acids and can provide insight into the H–D exchange reactions observed with polyprotonated proteins produced by electrospray ionization.