Synthesis and use of stereospecifically deuterated analogues of palmitic Acid to investigate the stereochemical course of the delta11 desaturase of the processionary moth

Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the immediate sex pheromone precursor. In this article, we describe the synthesis of different stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher the stereochemical course of the T. pityocampa Delta(11) desaturase. The synthesis of probes has been carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental evidence is presented that the Delta(11) desaturase of T. pityocampa transforms palmitic acid into (Z)-11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12.     

Molecular evolution of the plant ECERIFERUM1 and ECERIFERUM3 genes involved in aliphatic hydrocarbon production

The Arabidopsis ECERIFERUM1 (CER1) protein is a decarbonylase that converts fatty acid metabolites into alkanes. Alkanes are components of waxes in the plant cuticle, a waterproof barrier serving to protect land plants from both biotic and abiotic stimuli. CER1 enzymes can be used to produce alternative and sustainable hydrocarbons in eukaryotic systems. In this report we identified 193 CER1 and 128 CER3 sequences from 56 land plants respectively. CER1 and CER3 proteins have high amino acid similarity and both are involved in alkane synthesis in Arabidopsis. The common homologues of CER1 and CER3 genes were identified in three species of chlorophytes, which may be one of the earliest plant taxa that possess CER1 and CER3 genes. To facilitate potential applications, the 3-dimensional structure and conserved motifs of CER1 proteins were also characterized. CER1 and CER3 proteins are structurally similar, but CER1 proteins have more conserved histidine-containing motifs common to fatty acid hydroxylases and stearoyl-CoA desaturases. There was no significant loss or gain of protein motifs after ancient and recent duplications, suggesting that varied properties of CER1 proteins may be associated with less-conserved regions. Among 56 land plants, the codon-based assessments of selection modes revealed that neither entire proteins nor individual amino acids of CER1 proteins were significantly subjected to positive selection, indicating that CER1 proteins are highly conserved throughout evolution.     

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.     

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.     

Microsomal delta 9, delta 6 and delta 5 desaturase activities and liver membrane fatty acid profiles in alcohol-fed rats

In experimentally produced alcoholic fatty liver microsomal fatty acid composition was measured using gas chromatography. The results showed an increase in linoleic acid (18:2, n-6) and hexadecaenoic acid (22:6, n-3) and a decrease in arachidonic acid (20:4, n-6) in alcohol-fed rats. Using high performance liquid chromatographic separation of radiolabelled substrate and products, delta 9, delta 6 and delta 5 desaturase enzymes were assayed. The activity of delta 9 and delta 5 desaturase was decreased in alcohol-fed rats and delta 6 desaturase activity was similar in control and alcohol-fed groups. These results indicated there was no causal relationship between desaturase activity and membrane fatty acid changes. Increased amounts of eicosatrienoic acid (20:3, n-9) in rats fed less than 5% fat were observed in both control and alcohol-fed rats. The results indicated that essential fatty acid deficiency was not due to alcohol consumption.     

Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts

It is necessary to eliminate linoleic acid and allergenic arachins from peanuts for good health reasons. Virginia-type peanuts, harvested from plots treated with mineral salts combinations that mimic the subunit compositions of glutamate dehydrogenase (GDH) were analyzed for fatty acid and arachin compositions by HPLC and polyacrylamide gel electrophoresis, respectively. Fatty acid desaturase and arachin encoding mRNAs were analyzed by Northern hybridization using the homologous RNAs synthesized by peanut GDH as probes. There were 70?C80?% sequence similarities between the GDH-synthesized RNAs and the mRNAs encoding arachins, fatty acid desaturases, glutamate synthase, and nitrate reductase, which similarities induced permutation of the metabolic pathways at the mRNA level. Modeling of mRNAs showed there were 210, 3,150, 1,260, 2,520, and 4,200 metabolic permutations in the control, NPKS-, NS-, Pi-, NH₄Cl-, and PK-treated peanuts, respectively. The mRNA cross-talks decreased the arachin to almost zero percent in the NPKS- and PK-treated peanuts, and linoleate to ??18?% in the PK-treated peanut. The mRNA cross-talks may account for the vastly reported environmentally induced variability in the linoleate contents of peanut genotypes. These results have quantitatively unified molecular biology and metabolic pathways into one simple biotechnology for optimizing peanut quality and may encourage small-scale industry to produce arachin-free low linoleate peanuts.     

Biocatalytic Properties and Structural Analysis of Phloroglucinol Reductases

Phloroglucinol reductases (PGRs) are involved in anaerobic degradation in bacteria, in which they catalyze the dearomatization of phloroglucinol into dihydrophloroglucinol. We identified three PGRs, from different bacterial species, that are members of the family of NAD(P)H‐dependent short‐chain dehydrogenases/reductases (SDRs). In addition to catalyzing the reduction of the physiological substrate, the three enzymes exhibit activity towards 2,4,6‐trihydroxybenzaldehyde, 2,4,6‐trihydroxyacetophenone, and methyl 2,4,6‐trihydroxybenzoate. Structural elucidation of PGRcl and comparison to known SDRs revealed a high degree of conservation. Several amino acid positions were identified as being conserved within the PGR subfamily and might be involved in substrate differentiation. The results enable the enzymatic dearomatization of monoaromatic phenol derivatives and provide insight into the functional diversity that may be found in families of enzymes displaying a high degree of structural homology.     

Stereochemistry of a bifunctional dihydroceramide delta 4-desaturase/hydroxylase from Candida albicans; a key enzyme of sphingolipid metabolism

The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.     

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD‐containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2‐(2‐hydroxyethoxy)acetic acid. Such a facile cofactor‐independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks.     

Insights to Sequence Information of Polyphenol Oxidase Enzyme from Different Source Organisms

Polyphenol oxidases (PPOs) are widely distributed enzymes among animals, plants, bacteria, and fungi. PPOs often have significant role in many biologically essential functions including pigmentation, sclerotization, primary immune response, and host defense mechanisms. In the present study, forty-seven full-length amino acid sequences of PPO from bacteria, fungi, and plants were collected and subjected to multiple sequence alignment (MSA), domain identification, and phylogenetic tree construction. MSA revealed that six histidine, two phenylalanine, two arginine, and two aspartic acid residues were highly conserved in all the analyzed species, while a single cysteine residue was conserved in all the plant and fungal PPOs. Two major sequence clusters were constructed by phylogenetic analysis. One cluster was of the plant origin, whereas the other one was of the fungal and bacterial origin. Motif GGGMMGDVPTANDPIFWLHHCNVDRLWAVWQ was found in all the species of bacterial and fungus sources. In addition, seven new motifs which were unique for their group were also identified.     

Composition of the triacylglycerols of the seeds of some representatives of the family labiatae

The fatty acid compositions of five species and the compositions of the triacylglycerols of 22 species of the family labiatae have been studied for the first time. Octadeca-ε12,13-dienoic acid has been detected in five species. The typical compositions of the triacylglycerols differs from those of known plant oils with a similar set of fatty acids by the absence of triacylglycerols of the S₃ type and the presence of the S₂U type (0.1–1.6%). The main types are SU₂ (5–24%) and U₃ (74–95%). In a comparison of the position-species composition of the oils studied it was found that the oils of the plants of this family are distinguished by a greater diversity of species of triacylglycerols and also by the nature of the distribution of the unsaturated acyl residues between the 1,3- and 2-positions. In the majority of oils studied, the 2- position is enriched with the 18:1 acid, while the 18:2 acid is distributed predominantly in the 1,3- positions, and the nature of the distribution of the 18:3 acid is determined by its proportion in the total.     

Computational Identification of MicroRNAs and Their Targets in Perennial Ryegrass (Lolium perenne)

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.     

Detection Mechanism of Metallized Carbon Epoxy Oxidase Enzyme Based Sensors

The coenzyme FAD has been identified to play an important role in the detection mechanism of oxidase enzyme based biosensors. Incorporating FAD into the carbon composite improved sensitivity to H₂O₂ consequently increasing sensitivity to the respective analyte. The amount of active enzyme also increased thus enhancing the overall performance of the sensors. Polycarboxybetaine (PCB) has been used as a biocompatible membrane coating. The PCB coated sensors gave reproducible calibrations in protein solutions, which has been shown to be a valid protocol for testing biocompatibility. The importance of reporting selectivity in a manner which indicates the “fitness of purpose” of biosensors has been discussed.     

Visualization of phosphatidylcholine,lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry

The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.     

Effects of Dietary α-Linolenic Acid Treatment and the Efficiency of Its Conversion to Eicosapentaenoic and Docosahexaenoic Acids in Obesity and Related Diseases

The essential fatty acid alpha-linolenic acid (ALA) is present in high amounts in oils such as flaxseed, soy, hemp, rapeseed, chia, and perilla, while stearidonic acid is abundant in echium oil. ALA is metabolized to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by desaturases and elongases in humans. The conversion of ALA to EPA and DHA is limited, and these long-chain n−3 polyunsaturated fatty acids (PUFAs) are mainly provided from dietary sources (fish and seafood). This review provides an overview of studies that explored the effects of dietary supplementation with ALA in obesity and related diseases. The obesity-associated changes of desaturase and elongase activities are summarized, as they could influence the metabolic conversion of ALA. Generally, supplementation with ALA or ALA-rich oils leads to an increase in EPA levels and has no effect on DHA or omega-3 index. According to the literature data, stearidonic acid could enhance conversion of ALA to long-chain n−3 PUFA in obesity. Recent studies confirm that EPA and DHA intake should be considered as a primary dietary treatment strategy for improving the omega-3 index in obesity and related diseases.     

High-performance liquid chromatography of cardiolipin

Resolution of freshly prepared and of commercially available (degraded) samples of cardiolipin into 15-30 components has been accomplished by reversed-phase high-performance liquid chromatography using a 3-micron particulate Microsorb C18 column irrigated with linear gradients of acetonitrile--methanol--10 mM phosphate buffer pH 7.4. Selected resolved components were crystallized and characterized by infrared absorption spectra. Saponification of other components and identification of component fatty acids by reversed-phase high-performance liquid chromatography demonstrated the presence of ten fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4), with linoleic acid (18:2) identified in all resolved components. From fatty acid composition data it appears that several resolved fractions consist of single cardiolipin molecular species.     

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

A series of 18‐fluoro thiastearates were prepared and incubated with a yeast Δ9‐desaturating system. The relative efficiency of desaturase‐mediated sulfoxidation was monitored via ¹⁹F‐NMR analysis of the sulfoxide products, and a strong preference for oxo transfer to the S‐atom occupying the 9‐position was confirmed. The oxidation profile obtained in this manner matched that of analogous experiments with non‐fluorinated substrates. These results form the basis of a versatile ¹⁹F‐NMR‐based method for mapping the position of the putative diiron oxidant relative to substrate, and has potential application to the study of membrane‐bound desaturases in vitro.     

Discovery of a Single Monooxygenase that Catalyzes Carbamate Formation and Ring Contraction in the Biosynthesis of the Legonmycins

Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD‐dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so‐called legonmycins. By genome‐driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non‐ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time.