Fatty Acid Biosynthetic Pathway Research Articles

Fatty acids of plant vacuolar membrane lipids

Fatty acid (FA) composition of vacuolar membrane lipids from storage tissues of umbelliferous plants, viz., parsnip (Pastinaca sativa L.), parsley (Petroselinium crispum L.), and carrot (Daucus carota L.) is studied by gas-liquid chromatography and the FA biosynthetic pathways are considered. Vacuolar membrane lipids are characterized by high (78% of the total FA pool) content of unsaturated FA among which linoleic acid is predominant. Its content in vacuolar lipids of parsnip, parsley and carrot is 53.5, 55.1, and 54.9%, respectively. Parsnip and parsley vacuolar lipids contain large amounts of hexadecadienoic C16:2ω6 acid (8.0 and 4.6%, respectively). The content of α-linolenic acid in vacuolar lipids of tested plants varies from 4.8 to 7.3%. Palmitic acid (18.0–20.7%) predominates among saturated FA. High content of linoleic and hexadecadienoic acid in parsnip and parsley vacuolar lipids is suggestive of a crucial role of the microsomal ω6 fatty-acid desaturase fad2 gene in resistance and acclimation of plants to low temperatures.

A Key Role for Orphan Nuclear Receptor Liver Receptor Homologue-1 in Activation of Fatty Acid Synthase Promoter by Liver X Receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.

Coupling of the de Novo Fatty Acid Biosynthesis and Lipoylation Pathways in Mammalian Mitochondria

The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein lipoylation pathway.

IGF‐1 stimulates de novo fatty acid biosynthesis by Schwann cells during myelination

Schwann cell (SC) differentiation to the myelinating phenotype is characterized by the elaboration of a lipid-rich membrane and the expression of myelin-specific proteins. Insulin-like growth factor-1 (IGF-1) has been identified as a growth factor that stimulates the early events of myelination in SCs that signals via the PI3K/Akt pathway. Given the role of IGF-1 in promoting myelination, we performed studies to determine if the fatty acid biosynthetic pathway was a target of IGF-1 signaling in the formation of myelin membrane in dorsal root ganglion neuron/Schwann cell (DRG/SC) cocultures. We report that the fatty acid profile of lipid extracts of cocultures treated with IGF-1 match that reported for native myelin membrane by electrospray mass spectroscopy analysis. We also demonstrate de novo fatty acid biosynthesis in response to IGF-1 treatment in DRG/SC cocultures metabolically labeled with (13)C-acetate as a carbon source for fatty acid synthesis. Consistent with this finding, Western blot analysis of lysates from both cocultures and purified SCs reveal that IGF-1 stimulates two key fatty acid synthesizing enzymes. Additionally, we show that stimulation of fatty acid synthesizing enzymes is mediated by the PI3K/Akt signaling pathway. We also show that the fatty acid synthesizing enzymes and associated signaling pathways are elevated during the period of myelin membrane formation in sciatic nerve. Collectively, these findings demonstrate that IGF-1 plays an important regulatory function during myelin membrane formation.

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.

Control of Energy Homeostasis: Role of Enzymes and Intermediates of Fatty Acid Metabolism in the Central Nervous System

The regulation of energy homeostasis is critical for normal physiology and survival. Energy flux must be rigorously monitored and adjusted to ensure that fuel intake and expenditure remain within acceptable limits. The central nervous system (CNS) is, in large part, responsible for conducting this energy-monitoring function and for integrating the numerous inputs. It has become evident that neurons of the CNS monitor and respond to levels of metabolic intermediates that reflect peripheral energy status. Intermediates in the fatty acid biosynthetic pathway have been implicated as hypothalamic signaling mediators that sense and respond to changes in circulating fuels. Genetic and pharmacologic manipulation of the enzymes of fatty acid metabolism have led to the hypothesis that neuronal metabolic intermediates affect neural outputs that modify both feeding behavior and energy expenditure. This review focuses on the regulatory roles of these enzymes and intermediates in the regulation of food intake and energy balance.

New mutants of winter rapeseed (Brassica napus L.) with changed fatty acid composition

AbstractSeeds of the winter oilseed rape (Brassica napus L.) line PN 3756/93 were treated with ethyl methanesulphonate to induce mutations in the fatty acid biosynthetic pathway. The seed mutagenic treatment was repeated in the M2 generation. After treatments, individual seed and plant selections were made for changes in fatty acid composition during several generations of inbreeding. Self‐pollinated plants with changed fatty acid compositions were inbred to obtain genetically homozygous and stable mutant lines. Two mutants, M‐10453 and M‐10464, with increased levels of oleic acid (approximately 76%) and reduced linoleic and linolenic acid contents (8.5% and 7.5%, respectively) were selected. Gene or genes controlling desaturation of oleic acid were probably mutated in these plants. The third mutant, M‐681 had a very low linolenic acid content (approximately 2.6%) and increased linoleic acid content (approximately 26%). This would suggest the occurrence of mutations in genes controlling linoleic acid desaturation. The results of selection work during several generations showed that the environment had substantial influence on the composition of seed oil. This made the search for mutants with modify fatty acid compositions difficult. The induced mutants are not directly usable as new varieties, but can be used as parents in crosses for the development of high quality rapeseed varieties.

A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.

The Peroxisomal Acyl-CoA Thioesterase Pte1p from Saccharomyces cerevisiae Is Required for Efficient Degradation of Short Straight Chain and Branched Chain Fatty Acids

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

The role of the endocannabinoid system in the control of energy homeostasis

The endocannabinoid system has recently emerged as an important regulator of energy homeostasis, involved in the control of both appetite and peripheral fat metabolism. We briefly review current understanding of the possible sites of action and cellular mechanisms involved in the central appetitive and peripheral metabolic effects of endocannabinoids. Studies in our laboratory, using leptin-deficient obese rodents and CB1 cannabinoid receptor (CB1)-deficient mice, have indicated that endocannabinoids acting via CB1 are involved in the hunger-induced increase in food intake and are negatively regulated by leptin in brain areas involved in appetite control, including the hypothalamus, limbic forebrain and amygdala. CB1-/- mice are lean and are resistant to diet-induced obesity (DIO) despite similar energy intake to wild-type mice with DIO, suggesting that CB1 regulation of body weight involves additional peripheral targets. Such targets appear to include both adipose tissue and the liver. CB1 expressed in adipocytes has been implicated in the control of adiponectin secretion and lipoprotein lipase activity. Recent findings indicate that both endocannabinoids and CB1 are present in the liver and are upregulated in DIO. CB1 stimulation increases de novo hepatic lipogenesis through activation of the fatty acid biosynthetic pathway. Components of this pathway are also expressed in the hypothalamus where they have been implicated in the regulation of appetite. The fatty acid biosynthetic pathway may thus represent a common molecular target for the central appetitive and peripheral metabolic effects of endocannabinoids.

ApoA-I

See related article, pages 564–571 A link between homocysteine and atherothrombotic vascular disease was first suggested by McCully more than 30 years ago.1 The vascular lesions observed by McCully in children with inborn errors of homocysteine metabolism were fibrotic rather than lipid rich, suggesting the possibility that hyperhomocysteinemia may produce vascular pathology through a mechanism independent of altered lipid metabolism. Since that time, many epidemiological studies have confirmed that elevated plasma levels of total homocysteine (tHcy) are associated with an increased risk of coronary events, stroke, and venous thromboembolism.2–4 Most of the epidemiological data indicate that elevation of plasma tHcy is not associated with a significant change in plasma total cholesterol but is negatively associated with high density lipoprotein (HDL) cholesterol.5–7 An effect of homocysteine on HDL metabolism could be clinically important, because HDL protects from vascular disease not only by facilitating reverse cholesterol transport8 but also through its direct antiinflammatory properties.9 During the past decade, the development of animal models has led to rapid progress in defining the pathophysiological consequences of hyperhomocysteinemia in vivo. Experimental approaches to induce hyperhomocysteinemia in animals include the use of diets that are high in methionine and/or low in folate, as well as genetic approaches, such as the generation of knockout mice with targeted disruption of the cystathionine β-synthase ( Cbs ),10 methylene tetrahydrofolate reductase ( Mthfr ),11 or methionine synthase ( Mtr )12 genes. Like hyperhomocysteinemic humans, hyperhomocysteinemic animals develop endothelial dysfunction, hepatic lipid accumulation, and decreased plasma HDL cholesterol with minimal changes in plasma …

Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa

The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A DeltafabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Delta11 as the principal UFA, whereas the DeltafabA strain produced only 16:1Delta9. The double bond in the 16:1Delta9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The DeltafabADeltadesA double mutant required exogenous fatty acids for growth but the DeltafabAdesB double mutant did not. Exogenous stearate was converted to 18:1Delta9 and supported the growth of DeltafabADeltadesA double mutant. A DeltafabADeltadesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in DeltafabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Delta9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Delta9-desaturase whose expression is repressed by DesT.

Discovery of Aerobic Mechanisms for the Formation of Unsaturated Fatty Acids in Pseudomonas aeruginosa

Unsaturated fatty acids (UFA) are produced anaerobically, or by oxidative desaturation in the presence of oxygen. Anaerobic synthesis introduces the double bond into the growing acyl chain by the FabA dehydratase/isomerase component of the bacterial type II fatty acid biosynthetic pathway. A ΔfabA mutant of Pseudomonas aeruginosa grew without exogenous UFA but was a UFA auxotroph under anaerobic conditions. Wild-type cells produced C18:1 as the principal UFA, while the ΔfabA strain produced mainly C16:1. Two membrane proteins in P. aeruginosa, DesA and DesB, possessed the characteristic desaturase histidine clusters which are essential for catalysis. No fatty acid compositional changes were found in either ΔdesA or ΔdesB strains. ΔfabAΔdesA double mutant required UFA for growth but ΔfabAΔdesB double mutant did not. DesA expression rescued ΔfabAΔdesA strain from UFA auxotroph and elevated C16:1 production, indicating that DesA was responsible for C16:1 formation in ΔfabA strain. Acetate pulse-chase experiments demonstrated that in the ΔfabA strain the double bond in C16:1 was introduced after fatty acid biosynthesis. The C16:1 was located in the sn-2 position of phosphatidylethanolamine and the double bond was at the Δ9 position. Stearate supported the growth of ΔfabAΔdesA double mutant, and it was converted to C18:1 9, whereas wild type cell has C18:1 11. The expression of desB was induced by stearate, and repressed by oleate. Thus, P. aeruginosa has both an oxygen-dependent phospholipid desaturase (DesA) and an acy-CoA desaturase (DesB). (supported by NIH Grant GM34496)

Characterization of Pseudomonas aeruginosa DesT (PA4890), a transcriptional factor sensing long‐chain acyl‐CoAs

Cells regulate their membrane phospholipid biophysical properties by coordinated synthesis of saturated and unsaturated fatty acids. In bacteria, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalyzed by desaturases. In this study, we have identified and characterized a unique transcriptional repressor in Pseudomonas aeruginosa, DesT (PA4890), which regulates the expression of an acyl-CoA desaturase operon (desCB, PA4889 and PA4888). The desCB operon is located immediately upstream of desT and is transcribed to the opposite direction to desT. Both Affymetrix array and quantitative real-time PCR analyses demonstrated that desC and desB expression were strongly and selectively up-regulated in a ΔdesT deletion strain. The purified recombinant DesT bound to the desCB promoter region at two distinct sites with different affinities. The interaction of DesT with the high-affinity binding site (Kd = 0.35 ± 0.03 nM) was enhanced by unsaturated acyl-CoAs such as palmitoleoyl-CoA and oleoyl-CoA (Kd = 0.10 ± 0.01 nM), whereas saturated acyl-CoAs such as myristoyl-CoA and palmitoyl-CoA prevented DesT interaction with the high-affinity site. CoA, short-chain acyl-CoAs, free fatty acids and acyl-ACPs did not influence DesT-DNA interactions in vitro. Binding to the low-affinity site was not regulated. Accordingly, the expression of desB was up-regulated in cells grown in the presence of stearate and repressed when oleate was present. Thus, DesT senses the composition of the cellular acyl-CoA pool to either increase or decrease the expression of the acyl-CoA Δ9 desaturase system to adjust fatty acid metabolism to the composition of the exogenous fatty acid pool.

The Genomic Analysis of the Impact of Steroid Receptor Coactivators Ablation on Hepatic Metabolism

Members of the steroid receptor coactivator (SRC) family, which include SRC-1 (NcoA-1/p160), SRC-2(TIF2/GRIP1/NcoA-2) and SRC-3(pCIP/RAC3/ACTR/pCIP/ AIB1/TRAM1), are critical mediators of steroid receptor action. Gene ablation studies previously identified SRC-1 and SRC-2 as being involved in the control of energy homeostasis. A more precise identification of the molecular pathways regulated by these coactivators is crucial for understanding the role of steroid receptor coactivators in the control of energy homeostasis and obesity. A genomic approach using microarray analysis was employed to identify the subsets of genes that are altered in the livers of SRC-1-/-, SRC-2-/-, and SRC-3-/- mice. Microarray analysis demonstrates that gene expression changes are specific and nonoverlapping for each SRC member in the liver. The overall pattern of altered gene expressions in the SRC-1-/- mice was up-regulation, whereas SRC-2-/- mice showed an overall down-regulation. Several key regulatory enzymes of energy metabolism were significantly altered in the liver of SRC-2-/- mice, which are consistent with the prior observation that SRC-2-/- mice have increased energy expenditure. This study demonstrates that the molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway, whereas fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.

Functional characterization of front‐end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Expression of Δ6, Δ5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [ Glycine max (L.) Merrill] seeds

We report the production of arachidonic acid (ARA) in seeds of soybean [ Glycine max (L.) Merrill] by seed-specific expression of the genes encoding Δ6 desaturase, fatty acid elongase, and Δ5 desaturase from a filamentous fungus, Mortierella alpina 1S-4, as well as the down-regulation of an endogenous gene of soybean, Δ15 desaturase. Several fatty acids, γ-linolenic acid (GLA), eicosa-8, 11-dienoic acid (EDA), dihomo-γ-linolenic acid (DGLA), and ARA, which the host plant does not produce, accumulated up to 11.0 and 8.4% of the total fatty acids in the transgenic histodifferentiated somatic embryos and the transgenic mature seeds, respectively. The ability to synthesize these novel fatty acids was inherited to their progenies. These results indicate that metabolic engineering of the fatty acid biosynthetic pathway to produce desired very long-chain polyunsaturated fatty acids in oilseed crops is feasible.

FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis

Fatty acid synthase (FAS), a key enzyme of the fatty acid biosynthetic pathway, has been shown to be overexpressed in various types of human cancer and is, therefore, considered to be an attractive target for anticancer therapy. However, the exact mechanism of overexpression of the FAS gene in tumor cells is not well understood. In this report, we demonstrate that the expression of the tumor suppressor gene PTEN has a significant inverse correlation with FAS expression in the case of prostate cancer in the clinical setting, and inhibition of the PTEN gene leads to the overexpression of FAS in vitro. We also found that the combination of the expression status of these two genes is a better prognostic marker than either gene alone. Furthermore, our results indicate that the specific inhibition of FAS gene by siRNA leads to apoptosis of prostate tumor cells, and inhibition of PI 3-kinase pathway synergizes with FAS siRNA to enhance tumor cell death. These results provide a strong rationale for exploring the therapeutic use of an inhibitor of the PTEN signaling pathway in conjunction with the FAS siRNA to inhibit prostate tumor growth.

Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii : Discovery of the Betaine Lipid Synthase BTA1 Cr

Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase

Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several α-keto acids. In its functional form, it is bound covalently in an amide linkage to the ε-amino group of a conserved lysine residue of the “lipoyl bearing subunit,” resulting in a long “swinging arm” that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg −1, indicating a turnover number of ∼0.2 s −1, and the apparent K m values for octanoyl-ACP and apo-H protein are 10.2 ± 4.4 and 13.2 ± 2.9 μM, respectively.