acyl-CoA Synthetase Activity Research Articles

Reconfiguring the Escherichia coli Electron Transport Chain to Enhance trans-2-Decenoic Acid Production.

trans-2-Decenoic acid is a pivotal α,β-medium-chain unsaturated fatty acid that serves as an essential intermediary in the synthesis of 10-hydroxy-2-decenoic acid and various pharmaceutical compounds. Biosynthesis yield of trans-2-decenoic acid by decanoic acid has significantly improved in recent years; however, the oxidative stress of Escherichia coli at high fatty acid concentrations restricts the conversion rate. Here, we introduced a combination of rational design and metabolic rewiring of the E. coli electron transport chain (ETC) to improve trans-2-decenoic acid production. Overexpressing ubiquinone (UbQ) biosynthesis genes enhanced the expression of ETC complex III: UbQ to reduce reactive oxygen species (ROS) accumulation. Furthermore, applying rotenone to inhibit ETC complex I improved the electron transfer efficiency of complex II. The integration of Vitamin B5 and B2 into the fermentation process increased the activities of fatty acyl-CoA synthetase (MaMACS) and fatty acyl-CoA dehydrogenase (PpfadE). Finally, the constructed E. coli BL21(DE3)(ΔfadBJR/pCDFDuet-1-PpfadE-MaMACS/pRSFDuet-1-sumo-CtydiI-ubiI) strain exhibited a 51.50% decrease in ROS and a 93.33% enhancement in trans-2-decenoic acid yield, reaching 1.45 g/L after 66 h, which is the highest yield reported for flask fermentation. This study reports the feasibility of rewiring the ETC regulation and energy metabolism to improve α,β-UCA biosynthesis efficiency.

In Staphylococcus aureus, the acyl-CoA synthetase MbcS supports branched-chain fatty acid synthesis from carboxylic acid and aldehyde precursors.

In the human pathogen Staphylococcus aureus, branched-chain fatty acids (BCFAs) are the most abundant fatty acids in membrane phospholipids. Strains deficient for BCFAs synthesis experience auxotrophy in laboratory culture and attenuated virulence during infection. Furthermore, the membrane of S. aureus is among the main targets for antibiotic therapy. Therefore, determining the mechanisms involved in BCFAs synthesis is critical to manage S. aureus infections. Here, we report that the overexpression of SAUSA300_2542 (annotated to encode an acyl-CoA synthetase) restores BCFAs synthesis in strains lacking the canonical biosynthetic pathway catalyzed by the branched-chain α-keto acid dehydrogenase (BKDH) complex. We demonstrate that the acyl-CoA synthetase activity of MbcS activates branched-chain carboxylic acids (BCCAs), and is required by S. aureus to utilize the isoleucine derivative 2-methylbutyraldehyde to restore BCFAs synthesis in S. aureus. Based on the ability of some staphylococci to convert branched-chain aldehydes into their respective BCCAs and our findings demonstrating that branched-chain aldehydes are in fact BCFAs precursors, we propose that MbcS promotes the scavenging of exogenous BCCAs and mediates BCFA synthesis via a de novo alternative pathway.

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

Recombinant expression and functional characterization of FadD2 protein in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a crucial and highly destructive intracellular pathogen responsible for causing tuberculosis (TB). The emergence and dissemination of multi-drug resistant Mtb has further aggravated the TB crisis, leading to high mortality. Mtb FadD2 is a fatty acyl-coenzyme A (CoA) synthetase that modifies the cell envelope and plays an important role in reducing Mtb susceptibility to pyrazinoic acid (POA). However, the functional mechanism of Mtb FadD2 remains poorly understood. Here, we successfully expressed, purified and obtained monomeric FadD2 by using buffer (500 mM NaCl, 20 mM Tris-HCl, pH7.4 and 5 % glycerol). Palmitate was found to be the optimal substrate for FadD2. Fatty acyl-CoA synthetase activity reached maximum at 450 μM palmitate, and the Km value was 318.2 μM for palmitate. The results of mutation experiments indicated the critical role of T370 and K551 in the enzymatic activity of FadD2. Our work provides a guideline and concept for the development of novel drugs against Mtb.

Acyl-CoA synthetase expression in human skeletal muscle is reduced in obesity and insulin resistance.

Upon intramuscular entry, fatty acids are converted to amphiphatic fatty acyl-CoAs by action of the acyl-CoA synthetase (ACS) enzymes. While it has been reported that insulin resistant skeletal muscle shows an accumulation of fatty acyl-CoAs, the role of the enzymes which catalyze their synthesis is still sparsely studied in human muscle, in particular the influence of obesity, and insulin resistance. We analyzed muscle biopsies obtained from normal weight controls (n = 7, average BMI 24), males/females with obesity (n = 7, average BMI 31), and males/females with obesity and type 2 diabetes (T2D) (n = 7, average BMI 34), for relevant ACS (long-chain acyl-CoA synthetase 1 (ACSL1), -3 (ACSL3) and - 4 (ACSL4), fatty acid transport protein 1 (FATP1) and - 4 (FATP4)). The mRNA expression was determined by real-time PCR, and total oleoyl-CoA synthetase activity was measured. In the males/females with obesity and T2D, the response to 16 weeks of exercise training with minor weight loss was evaluated. ACSL1 is the dominantly expressed ACS isoform in human skeletal muscle. The content of total ACS mRNA, as well as ACSL1 mRNA, were lower in muscle of males/females with obesity and T2D. Exercise training in the males/females with obesity and T2D increased the total ACS enzyme activity, along with a lowering of the HOMA-IR index. The capacity for synthesis of fatty acyl-CoAs is lower in skeletal muscle of obese males/females with T2D. This suggests a decreased ability to convert fatty acids to fatty acyl-CoAs, which in turn may affect their entry into storage or metabolic pathways in muscle. Thus, the accumulation of fatty acyl-CoAs in the obese or insulin resistant state that has been shown in previous reports is not likely to result from increased fatty acid acylation. The upregulation of ACS activity by exercise training appears beneficial and occurred concomitantly with increased insulin sensitivity.

The oleaginous yeast Starmerella bombicola reveals limitations of Saccharomyces cerevisiae as a model for fatty acid transport studies.

Saccharomyces cerevisiae is the model organism to most yeast researchers, and information obtained from its physiology is generally extrapolated to other yeasts. Studies on fatty acid transport in S. cerevisiae are based on the expression of both native fatty acid export genes as well as heterologous proteins. Starmerella bombicola, on the other hand, is an oleaginous yeast of industrial relevance but its fatty acid transport mechanisms are unknown. In this study, we attempt to use existing knowledge from S. cerevisiae to study fatty acid transport in S. bombicola, but the obtained results differ from those observed in S. cerevisiae. First, we observed that deletion of SbPRY1 in S. bombicola leads to higher fatty acid export, the opposite effect to the one previously observed for the Pry homologues in S. cerevisiae. Second, following reports that human FATP1 could export fatty acids and alcohols in S. cerevisiae, we expressed FATP1 in a fatty acid-accumulating S. bombicola strain. However, FATP1 reduced fatty acid export in S. bombicola, most likely due to its acyl-CoA synthetase activity. These results not only advance knowledge on fatty acid physiology of S. bombicola, but also improve our understanding of S. cerevisiae and its limitations as a model organism.

An Atypical Acyl-CoA Synthetase Enables Efficient Biosynthesis of Extender Units for Engineering a Polyketide Carbon Scaffold.

Acyl-CoAs are key precursors of primary and secondary metabolism. Their efficient biosynthesis is often impeded by the limited substrate specificity and low in vivo activity of acyl-CoA synthetases (ACSs) due to regulatory acylation of the catalytically important lysine residue in motif A10 (Lys-A10). In this study, we identified an unusual ACS (UkaQ) from the UK-2A biosynthetic pathway that naturally lacks the Lys-A10 residue and exhibits extraordinarily broad substrate specificity. Protein engineering significantly improved its stability and catalytic activity, enabling it to synthesize a large variety of acyl-CoAs with highly robust activity. By combining it with permissive carboxylases, we produced a large array of polyketide extender units and obtained six novel halobenzyl-containing antimycin analogues through an engineered biosynthetic pathway. This study significantly expands the catalytic mode of ACSs and provides a potent tool for the biosynthesis of acyl-CoA-derived natural products.

Functional Analysis of an Acyltransferase-Like Domain from Polyunsaturated Fatty Acid Synthase in Thraustochytrium.

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5-4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (∆fabDts) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.

Modulation of Gene Expression in Actinobacteria by Translational Modification of Transcriptional Factors and Secondary Metabolite Biosynthetic Enzymes.

Different types of post-translational modifications are present in bacteria that play essential roles in bacterial metabolism modulation. Nevertheless, limited information is available on these types of modifications in actinobacteria, particularly on their effects on secondary metabolite biosynthesis. Recently, phosphorylation, acetylation, or phosphopantetheneylation of transcriptional factors and key enzymes involved in secondary metabolite biosynthesis have been reported. There are two types of phosphorylations involved in the control of transcriptional factors: (1) phosphorylation of sensor kinases and transfer of the phosphate group to the receiver domain of response regulators, which alters the expression of regulator target genes. (2) Phosphorylation systems involving promiscuous serine/threonine/tyrosine kinases that modify proteins at several amino acid residues, e.g., the phosphorylation of the global nitrogen regulator GlnR. Another post-translational modification is the acetylation at the epsilon amino group of lysine residues. The protein acetylation/deacetylation controls the activity of many short and long-chain acyl-CoA synthetases, transcriptional factors, key proteins of bacterial metabolism, and enzymes for the biosynthesis of non-ribosomal peptides, desferrioxamine, streptomycin, or phosphinic acid-derived antibiotics. Acetyltransferases catalyze acetylation reactions showing different specificity for the acyl-CoA donor. Although it functions as acetyltransferase, there are examples of malonylation, crotonylation, succinylation, or in a few cases acylation activities using bulky acyl-CoA derivatives. Substrates activation by nucleoside triphosphates is one of the central reactions inhibited by lysine acetyltransferases. Phosphorylation/dephosphorylation or acylation/deacylation reactions on global regulators like PhoP, GlnR, AfsR, and the carbon catabolite regulator glucokinase strongly affects the expression of genes controlled by these regulators. Finally, a different type of post-translational protein modification is the phosphopantetheinylation, catalized by phosphopantetheinyl transferases (PPTases). This reaction is essential to modify those enzymes requiring phosphopantetheine groups like non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. Up to five PPTases are present in S. tsukubaensis and S. avermitilis. Different PPTases modify substrate proteins in the PCP or ACP domains of tacrolimus biosynthetic enzymes. Directed mutations of genes encoding enzymes involved in the post-translational modification is a promising tool to enhance the production of bioactive metabolites.

SYNTHESIS OF PROBE MOLECULES, 6-(DIMETHYLAMINO)-2-PHENYLISOINDOLIN-1-ONES, FOR MECHANISTIC STUDIES OF FIREFLY LUCIFERASE INHIBITION.

Firefly luciferase is used in high-throughput screening based on the detection of chemiluminescence. It catalyzes an esterification reaction of luciferin with adenosine 5'-triphosphate (ATP) followed by decarbonylation with oxygen and concomitance of light. Previously, we reported that firefly luciferase also possesses acyl-CoA synthetase activity and catalyzes an aromatic carboxylic acid group of F-53, using ATP, Mg2+ and coenzyme A (CoA), to produce F-53 covalently attached to active-site lysine-529 residue of firefly luciferase through the formation of an amide group. The amidation of lysine-529 resulted in a deactivation of luciferase. In order to probe firefly luciferase inhibition's mechanism, we synthesized two probe molecules 1 and 2, mimicking F-53. Molecule 1 contains an azido-appended side chain in the aromatic ring of F-53, while 2 possesses an azido and a carboxylic acid group appended side chains. Both synthetic schemes are readily amenable to large-scale syntheses. Molecule 1 was made from 2-allylaniline, which was derived from a thermal-induced aromatic-Claisen rearrangement of N-allylaniline. The azido-appended side chain of 2 was installed from a Horner-Wadsworth-Emmons reaction and the carboxylic acid side chain from a Sonogashira reaction.

Impaired mitochondrial medium-chain fatty acid oxidation drives periportal macrovesicular steatosis in sirtuin-5 knockout mice

Medium-chain triglycerides (MCT), containing C8–C12 fatty acids, are used to treat several pediatric disorders and are widely consumed as a nutritional supplement. Here, we investigated the role of the sirtuin deacylase Sirt5 in MCT metabolism by feeding Sirt5 knockout mice (Sirt5KO) high-fat diets containing either C8/C10 fatty acids or coconut oil, which is rich in C12, for five weeks. Coconut oil, but not C8/C10 feeding, induced periportal macrovesicular steatosis in Sirt5KO mice. 14C–C12 degradation was significantly reduced in Sirt5KO liver. This decrease was localized to the mitochondrial β-oxidation pathway, as Sirt5KO mice exhibited no change in peroxisomal C12 β-oxidation. Endoplasmic reticulum ω-oxidation, a minor fatty acid degradation pathway known to be stimulated by C12 accumulation, was increased in Sirt5KO liver. Mice lacking another mitochondrial C12 oxidation enzyme, long-chain acyl-CoA dehydrogenase (LCAD), also developed periportal macrovesicular steatosis when fed coconut oil, confirming that defective mitochondrial C12 oxidation is sufficient to induce the steatosis phenotype. Sirt5KO liver exhibited normal LCAD activity but reduced mitochondrial acyl-CoA synthetase activity with C12. These studies reveal a role for Sirt5 in regulating the hepatic response to MCT and may shed light into the pathogenesis of periportal steatosis, a hallmark of human pediatric non-alcoholic fatty liver disease.

A Vectorial, ER-Mitochondria Link to Energy Homeostasis in the Vascular Endothelium

The precise mechanisms of free fatty acid (FFA) uptake in the vascular endothelium are unclear. In this issue of Cell Metabolism, Ibrahim etal. (2020) discover that FFA uptake is partially mediated by a vectorial, ER-mitochondria link, in which mitochondrial ATP production is locally used for the acyl-CoA synthetase activity of the ER-located fatty acid transport protein 4.

An apple long-chain acyl-CoA synthetase 2 gene enhances plant resistance to abiotic stress by regulating the accumulation of cuticular wax.

Apple cuticular wax can protect plants from environmental stress, determine fruit luster and improve postharvest fruit storage quality. In recent years, dry weather, soil salinization and adverse environmental conditions have led to declines in apple fruit quality. However, few studies have reported the molecular mechanisms of apple cuticular wax biosynthesis. In this study, we identified a long-chain acyl-CoA synthetase MdLACS2 gene from apple. The MdLACS2 protein contained an AMP-binding domain and demonstrated long-chain acyl-CoA synthetase activity. MdLACS2 transgenic Arabidopsis exhibited reductions in epidermal permeability and water loss; change in the expression of genes related to cuticular wax biosynthesis, transport and transcriptional regulation; and differences in the composition and ultrastructure of cuticular wax. Moreover, the accumulation of cuticular wax enhanced the resistance of MdLACS2 transgenic plants to drought and salt stress. The main protein functional interaction networks of LACS2 were predicted, revealing a preliminary molecular regulation pathway for MdLACS2-mediated wax biosynthesis in apple. Our study provides candidate genes for breeding apple varieties and rootstocks with better fruit quality and higher stress resistance.

Local Mitochondrial ATP Production Regulates Endothelial Fatty Acid Uptake and Transport

Most organs use fatty acids (FAs) as a key nutrient, but little is known of how blood-borne FAs traverse the endothelium to reach underlying tissues. We conducted a small-molecule screen and identified niclosamide as a suppressor of endothelial FA uptake and transport. Structure/activity relationship studies demonstrated that niclosamide acts through mitochondrial uncoupling. Inhibitors of oxidative phosphorylation and the ATP/ADP translocase also suppressed FA uptake, pointing principally to ATP production. Decreasing total cellular ATP by blocking glycolysis did not decrease uptake, indicating that specifically mitochondrial ATP is required. Endothelial FA uptake is promoted by fatty acid transport protein 4 (FATP4) via its ATP-dependent acyl-CoA synthetase activity. Confocal microscopy revealed that FATP4 resides in the endoplasmic reticulum (ER), and that endothelial ER is intimately juxtaposed with mitochondria. Together, these data indicate that mitochondrial ATP production, but not total ATP levels, drives endothelial FA uptake and transport via acyl-CoA formation in mitochondrial/ER microdomains.

An apple long-chain acyl-CoA synthetase, MdLACS4, induces early flowering and enhances abiotic stress resistance in Arabidopsis

The aerial parts of apple are protected against environmental stress by cuticular wax. Although it has been suggested that several long-chain acyl-CoA synthetases are involved in wax biosynthesis, the molecular pathway of apple cuticular wax biosynthesis remains unclear. In this study, an MdLACS4 protein with long-chain acyl-CoA synthetase activity was isolated from apple. The MdLACS4 gene was highly expressed in pericarp, stem, and mature leaf tissues. Ectopic expression of MdLACS4 in Arabidopsis induced early flowering. Compared with wild-type plants, MdLACS4 transgenic Arabidopsis exhibited lower water loss rates, reduced epidermal permeability, increased cuticular wax in stems and leaves, and altered cuticular ultrastructure. Furthermore, the accumulation of cuticular wax enhanced the resistance of MdLACS4 transgenic plants to drought and salt stress. Finally, predicted protein functional interaction networks for LACS4 suggested that the molecular regulation pathway of MdLACS4 mediates wax biosynthesis in apple.

Acyl-CoA Synthetase Inhibition Protects Clonal Pancreatic Beta-cell from Effects of Chronic Excess Nutrients

Abstract Objectives A single nucleotide polymorphism (SNP) in the transcription factor 7 like 2 (TCF7L2) gene is strongly associated with Type 2 Diabetes (T2D), and deletion of this SNP has been shown to reduce long chain acyl-CoA synthetase 5 (ACSL5) mRNA level. Previous research in our lab has shown chronic exposure to excess nutrients (glucose and fatty acid (FA)) increases lipid droplets in beta-cells and causes basal insulin hypersecretion, left-shifted glucose-stimulated insulin secretion (GSIS), blunted maximal GSIS and reduced insulin content. We tested the efficacy of a known phenylpyrazole-carboxamide compound (herein named ADIPO C) to reduce accumulation of intracellular lipid droplets and reverse the left-shift of GSIS in beta-cells. Methods INS-1832/13 cells were cultured in RPMI media containing 10% FBS (source of FA) and either 4 or 11 mM glucose. Insulin was measured by HTRF assay (CisBio). Intracellular lipid was detected by fluorescence microscopy using Nile red. Fluorescent Bodipy-FA was used as a surrogate FA in both ACS activity assays and lipid incorporation into cells. Oxygen consumption rate was measured using the Seahorse ion flux analyzer and intracellular calcium was measured in fura 2 loaded cells mounted on an Olympus confocal microscope. Results Adipo C (10–25 µM) acutely inhibited acyl-CoA synthetase activity by up to 40% in cell homogenates and similarly reduced FA incorporation into neutral lipids in INS-1 (832/13) cells measured using thin layer chromatography. Longer exposure (72 hrs) to ADIPO C significantly decreased intracellular lipid droplets, right-shifted GSIS and increased insulin content in cells cultured in excess nutrients. Cells cultured in excess nutrients exhibited both increased basal oxygen consumption rate and intracellular calcium oscillations, which were both reduced with 72 hrs ADIPO C incubation. Conclusions Based on these results, we conclude that Adipo C has a protective effect on beta-cells exposed to a glucolipotoxic environment and thus may prove to have therapeutic potential in the prevention/treatment of T2D. Funding Sources No funding sources to report.

Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid ω-O-acylceramide

The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to ∼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.

Enzymatic promiscuity and the evolution of bioluminescence.

Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases and luciferins independently, all of which accomplish the impressive feat of light emission. One of the best-known examples of bioluminescence is exhibited by fireflies, a class of beetles that use d-luciferin as their substrate. The evolution of bioluminescence in beetles is thought to have emerged from ancestral fatty acyl-CoA synthetase (ACS) enzymes present in all insects. This theory is supported by multiple lines of evidence: Beetle luciferases share high sequence identity with these enzymes, often retain ACS activity, and some ACS enzymesfrom nonluminous insects can catalyze bioluminescence from synthetic d-luciferin analogues. Recent sequencing of firefly genomes and transcriptomes further illuminates how the duplication of ACS enzymesand subsequent diversification drove the evolution of bioluminescence. These genetic analyses have also uncovered candidate enzymes that may participate in luciferin metabolism. With the publication of the genomes and transcriptomes of fireflies and related insects, we are now better positioned to dissect and learn from the evolution of bioluminescence in beetles.

Acyl-CoA synthetases from Physcomitrella, rice and Arabidopsis: different substrate preferences but common regulation by MS188 in sporopollenin synthesis.

ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.

Defective fatty acid oxidation in mice with muscle-specific acyl-CoA synthetase 1 deficiency increases amino acid use and impairs muscle function

Loss of long-chain acyl-CoA synthetase isoform-1 (ACSL1) in mouse skeletal muscle (Acsl1M-/-) severely reduces acyl-CoA synthetase activity and fatty acid oxidation. However, the effects of decreased fatty acid oxidation on skeletal muscle function, histology, use of alternative fuels, and mitochondrial function and morphology are unclear. We observed that Acsl1M-/- mice have impaired voluntary running capacity and muscle grip strength and that their gastrocnemius muscle contains myocytes with central nuclei, indicating muscle regeneration. We also found that plasma creatine kinase and aspartate aminotransferase levels in Acsl1M-/- mice are 3.4- and 1.5-fold greater, respectively, than in control mice (Acsl1flox/flox ), indicating muscle damage, even without exercise, in the Acsl1M-/- mice. Moreover, caspase-3 protein expression exclusively in Acsl1M-/- skeletal muscle and the presence of cleaved caspase-3 suggested myocyte apoptosis. Mitochondria in Acsl1M-/- skeletal muscle were swollen with abnormal cristae, and mitochondrial biogenesis was increased. Glucose uptake did not increase in Acsl1M-/- skeletal muscle, and pyruvate oxidation was similar in gastrocnemius homogenates from Acsl1M-/- and control mice. The rate of protein synthesis in Acsl1M-/- glycolytic muscle was 2.1-fold greater 30 min after exercise than in the controls, suggesting resynthesis of proteins catabolized for fuel during the exercise. At this time, mTOR complex 1 was activated, and autophagy was blocked. These results suggest that fatty acid oxidation is critical for normal skeletal muscle homeostasis during both rest and exercise. We conclude that ACSL1 deficiency produces an overall defect in muscle fuel metabolism that increases protein catabolism, resulting in exercise intolerance, muscle weakness, and myocyte apoptosis.