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Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.

Medium-chain acyl-coenzyme-A dehydrogenase (MCAD) catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs. Mutations in the ACADM gene cause MCAD deficiency presenting with life-threatening symptoms during catabolism. Since fatty-acid-oxidation disorders are part of newborn screening (NBS), many novel mutations with unknown clinical relevance have been identified in asymptomatic newborns. Eighteen of these mutations were separately cloned into the human ACADM gene, heterologously overexpressed in Escherichia coli and functionally characterized by using different substrates, molecular chaperones, and measured at different temperatures. In addition, they were mapped to the three-dimensional MCAD structure, and cross-link experiments were performed. This study identified variants that only moderately affect the MCAD protein in vitro, such as Y42H, E18K, and R6H, in contrast to the remaining 15 mutants. These three mutants display residual octanoyl-CoA oxidation activities in the range of 22 % to 47 %, are as temperature sensitive as the wild type, and reach 100 % activity with molecular chaperone co-overexpression. Projection into the three-dimensional protein structure gave some indication as to possible reasons for decreased enzyme activities. Additionally, six of the eight novel mutations, functionally characterized for the first time, showed severely reduced residual activities < 5 % despite high expression levels. These studies are of relevance because they classify novel mutants in vitro on the basis of their corresponding functional effects. This basic knowledge should be taken into consideration for individual management after NBS.

Microbial fermentation is emerging as an increasingly important resource for the production of fatty acids to serve as precursors for renewable diesel as well as detergents, lubricants and other industrial chemicals, as an alternative to traditional sources of reduced carbon such as petroleum. A major disadvantage of fuels derived from biological sources is their undesirable physical properties such as high cloud and pour points, and high viscosity. Here we report the development of an Escherichia coli strain that efficiently produces anteiso-branched fatty acids, which can be converted into downstream products with lower cloud and pour points than the mixtures of compounds produced via the native metabolism of the cell. This work addresses a serious limitation that must be overcome in order to produce renewable biodiesel and oleochemicals that perform as well as their petroleum-based counterparts., (Published by Elsevier Inc.)

5-Aminolevulinate (ALA), an essential metabolite in all heme-synthesizing organisms, results from the pyridoxal 5'-phosphate (PLP)-dependent enzymatic condensation of glycine with succinyl-CoA in non-plant eukaryotes and α-proteobacteria. The predicted chemical mechanism of this ALA synthase (ALAS)-catalyzed reaction includes a short-lived glycine quinonoid intermediate and an unstable 2-amino-3-ketoadipate intermediate. Using liquid chromatography coupled with tandem mass spectrometry to analyze the products from the reaction of murine erythroid ALAS (mALAS2) with O-methylglycine and succinyl-CoA, we directly identified the chemical nature of the inherently unstable 2-amino-3-ketoadipate intermediate, which predicates the glycine quinonoid species as its precursor. With stopped-flow absorption spectroscopy, we detected and confirmed the formation of the quinonoid intermediate upon reacting glycine with ALAS. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine was reacted with ALAS, a lag phase was observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in ALAS. Hysteresis was not detected in the T148A-catalyzed L-serine external aldimine formation. These results with T148A, a mALAS2 variant, which, in contrast to wild-type mALAS2, is active with L-serine, suggest that active site Thr-148 modulates ALAS strict amino acid substrate specificity. The rate of ALA release is also controlled by a hysteretic kinetic mechanism (observed as a lag in the ALA external aldimine formation progress curve), consistent with conformational changes governing the dissociation of ALA from ALAS., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.

This study investigated the protective properties of garlic essential oil (GEO) and its major organosulfur component (diallyl disulfide, DADS) against the development of nonalcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a normal or high-fat diet (HFD) with/without GEO (25, 50, and 100 mg/kg) or DADS (10 and 20 mg/kg) for 12 weeks. GEO and DADS dose-dependently exerted antiobesity and antihyperlipidemic effects by reducing HFD-induced body weight gain, adipose tissue weight, and serum biochemical parameters. Administration of 50 and 100 mg/kg GEO and 20 mg/kg DADS significantly decreased the release of pro-inflammatory cytokines in liver, accompanied by elevated antioxidant capacity via inhibition of cytochrome P450 2E1 expression during NAFLD development. The anti-NAFLD effects of GEO and DADS were mediated through down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as stimulation of peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase-1. These results demonstrate that GEO and DADS dose-dependently protected obese mice with long-term HFD-induced NAFLD from lipid accumulation, inflammation, and oxidative damage by ameliorating lipid metabolic disorders and oxidative stress. The dose of 20 mg/kg DADS was equally as effective in preventing NAFLD as 50 mg/kg GEO containing the same amount of DADS, which demonstrates that DADS may be the main bioactive component in GEO.

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein-protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background & Aims: Diets with low omega (ω)-6 polyunsaturated fatty acids (PUFA) to eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) ratios have been shown to decrease aortic cholesterol accumulation and have been suggested to promote weight loss. The involvement of the liver and gonadal adipose tissue (GAT) in mediating these effects is not well understood. LDL receptor null mice were used to assess the effect of an atherogenic diet with different ω-6:EPA+DHA ratios on weight gain, hepatic and GAT lipid accumulation, and their relationship to atherosclerosis., Methods: Four groups of mice were fed a high saturated fat and cholesterol diet (HSF ω-6) alone, or with ω-6 PUFA to EPA+DHA ratios up to 1:1 for 32 weeks. Liver and GAT were collected for lipid and gene expression analysis., Results: The fatty acid profile of liver and GAT reflected the diets. All diets resulted in similar weight gains. Compared to HSF ω-6 diet, the 1:1 ratio diet resulted in lower hepatic total cholesterol (TC) content. Aortic TC was positively correlated with hepatic and GAT TC and triglyceride. These differences were accompanied by significantly lower expression of CD36, ATP-transporter cassette A1, scavenger receptor B class 1, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), acetyl-CoA carboxylase alpha, acyl-CoA synthetase long-chain family member 5, and stearoyl-coenzyme A desaturase 1 (SCD1) in GAT, and HMGCR, SCD1 and cytochrome P450 7A1 in liver., Conclusions: Dietary ω-6:EPA+DHA ratios did not affect body weight, but lower ω-6:EPA+DHA ratio diets decreased liver lipid accumulation, which possibly contributed to the lower aortic cholesterol accumulation., (Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Fatty acid short-chain esters (FASEs) are biodiesels that are renewable, nontoxic, and biodegradable biofuels. A novel approach for the biosynthesis of FASEs has been developed using metabolically-engineered E. coli through combination of the fatty acid and 2-keto acid pathways. Several genetic engineering strategies were also developed to increase fatty acyl-CoA availability to improve FASEs production. Fed-batch cultivation of the engineered E. coli resulted in a titer of 1008 mg/L FASEs. Since the fatty acid and 2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in a variety of microorganisms to produce various FASEs from cheap and readily-available, renewable, raw materials such as sugars and cellulose in the future., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Asthma is the most common chronic childhood disease. Imbalance of cytokines released from T helper cells and environmental factors, such as exposure to poly-aromatic hydrocarbon (PAH), play pivotal roles in the pathogenesis of asthma. The aim of this study was to compare the mRNA expression patterns of Interleukin (IL)-4, interferon (IFN)-γ and Acyl Co A long chain 3 (ACSL3) in peripheral blood leukocytes of children with and without asthma. To correlate the obtained mRNA data with serum IL-4, IFN-γ and PAH levels. Further, to determine the effect of in vivo exposure to PAH on mRNA expression of IL-4, IFN-γ and ACSL3 genes in a rat model., Methods: A total of 170 children below 16 years (85 pediatric asthma patients and 85 matched healthy controls) were randomly selected from the Riyadh Cohort, Saudi Arabia. Gene expression analysis was performed using qRTPCR. Serum IL-4, IFN-γ and PAH were measured using LINCOplex (human multiplex immunoassay kit) and HPLC respectively., Results: IL-4 mRNA expression was significantly increased (P < 0.05) in children with asthma compared to healthy control group whereas no differences were observed for either IFN-γ or ACSL3 mRNA. Similarly, serum IL- 4 and PAHs concentration was significantly higher as well in children with asthma in whom IFN-γ was also significantly lower. Results obtained in rats showed that exposure to the benzopyrene prototype PAH resulted in a 2.6 fold (P < 0.001) increased IL-4 mRNA expression in blood., Conclusion: Peripheral blood IL-4 mRNA levels, serum concentration of this cytokine are elevated in children with asthma. Also, elevated levels of PAH were observed in children with asthma. Additionally, PAH administration in rodents resulted in an increased IL-4 mRNA which is supposed to partly mediate the inflammatory response noted in asthma.

We provide novel insights into the function(s) of β-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving β-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue., (© 2013.)

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4-6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.

Background: Sam-Hwang-Sa-Sim-Tang (SHSST) is a traditional Oriental medication that has been commonly used in Korea for the treatment of hypertension, insomnia, and chest pain. In addition, some studies reported that administration of SHSST results suppression of hyperlipidemia in rats or lowering lipid plasma level such as total cholesterol (TC). Those results made us find and demonstrate positive effect of SHSST much more. The aim of the current study was to examine whether SHSST exerts an effect against hepatic steatosis and two type of SHSST has different efficacy on liver steatosis., Methods: Total 40 mice were divided randomly and equally into 4 groups: a normal diet (CON) group, high-cholesterol diet (HC) group, and treatment groups fed a high-cholesterol diet (HCD) with a 30% or 80% ethanol extract of SHSST (SHSST-L and SHSST-H, respectively). The HCD was given for 9 weeks. The SHSST-treated groups were orally administered SHSST at a dose of 150 mg/kg, whereas the other groups received physiological saline., Results: SHSST administration to mice resulted in a decline in serum levels of total cholesterol and low-density lipoprotein. Histological examination showed that lipid droplets were smaller in the SHSST-treated group than in the HC group. At the protein level, expression of sterol regulatory element-binding protein 2 (SREBP-2) was suppressed by SHSST. In addition, the mRNA expression of cholesterol metabolism-related molecules such as SREBP-2, liver X receptor (LXR), low-density lipoprotein receptor (LDLR), and 3-hydroxy-3methylglutary-CoA (HMG-CoA) was also suppressed in SHSST-treated groups in the liver. In the aorta tissue, SHSST decreased the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), transforming growth factor (TGF)-β1, and fibronectin., Conclusions: The present study indicates that SHSST protects against liver steatosis and protects vessels against inflammation arising from excessive ingestion of cholesterol. These findings may also suggest that SHSST could be used as an adjuvant remedy for protection against liver steatosis.

The major aim of this study is to elucidate the hypocholesterolemic mechanism exerted by rice protein (RP) in adult rats under cholesterol-enriched dietary condition. Compared with casein, the cholesterol levels in plasma and the liver were significantly reduced by RP, accompanying significant inhibition of cholesterol absorption. RP increased the activity and mRNA level of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase activity and gene expression were significantly depressed with consumption of RP. Neither the activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP differed from that of casein. The gene expression of the peroxisome proliferator-activated receptor α and liver X receptor α were significantly activated by consumption of RP. RP did not modify the mRNA level of sterol regulatory element-binding protein-2 with respect to casein. These results suggest RP can induce a cholesterol-lowering effect through modifying cholesterol metabolism-related gene expression and enzyme activity in adult rats.

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.

Objective: The aim of this study was to explore the genetic features of a family with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MHBDD) which may provide the basis for the diagnosis and genetic counseling., Method: Clinical data of the proband was collected, total RNA and genomic DNA were extracted from the peripheral blood. The whole coding region of the ACAT1 gene was amplified by RT-PCR. 5' noncoding region of the ACAT1 gene and all 6 exons and flanking intron regions of the HADH2 gene were amplified by PCR. All amplification products were directly sequenced and compared with the reference sequence., Result: (1) The patient was a one-year-old boy who presented with psychomotor retardation and astasia when he was admitted to the hospital. Biochemical test revealed slight hyperlactatemia (3.19 mmol/L) and magnetic resonance imaging showed delayed myelination. 2-Methylacetoacetyl-CoA thiolase deficiency was suggested by gas chromatography-mass spectrometry. (2) There was no mutation in the ACAT1 gene and a hemizygous missense mutation c.388C > T was found in the 4 exon of the HADH2 gene which resulted in p. R130C. Proband's mother was the heterozygote and the father was normal., Conclusion: This is the first report on MHBDD patient and HADH2 mutation in China. p.R130C is responsible for the pathogenesis of the disease in the infant.

The heterologous production of the complex antibiotic erythromycin through Escherichia coli provides a unique challenge in metabolic engineering. In addition to introducing the 19 foreign genes needed for heterologous biosynthesis, E. coli metabolism must be engineered to provide the propionyl-CoA and (2S)-methylmalonyl-CoA substrates required to allow erythromycin formation. In this work, three different pathways to propionyl-CoA were compared in the context of supporting E. coli erythromycin biosynthesis. The comparison revealed that alternative citramalate and threonine metabolic pathways (both starting from exogenous glycerol) were capable of supporting final compound formation equal to a proven pathway reliant upon exogenous propionate. Furthermore, two pathways to (2S)-methylmalonyl-CoA were compared in the production of a novel benzyl-erythromycin analog. A pathway dependent upon exogenous methylmalonate improved selectivity and facilitated antibiotic assessment of this new analog., (© 2013 Elsevier Inc. All rights reserved.)

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.

In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation-reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations. With a focus in this publication on the first hydride transfer, our structures follow this reduction reaction as the enzyme converts the HMG-CoA thioester from a flat sp(2)-like geometry to a pyramidal thiohemiacetal configuration consistent with a transition to an sp(3) orbital. This change in the geometry propagates through the coenzyme A (CoA) ligand whose first amide bond is rotated 180° where it anchors a web of hydrogen bonds that weave together the nucleotide, the reaction intermediate, the enzyme, and the catalytic residues. This creates a stable intermediate structure prepared for nucleotide exchange and the second reduction reaction within the HMG-CoA reductase active site. Identification of this reaction intermediate provides a template for the development of an inhibitor that would act as an antibiotic effective against the HMG-CoA reductase of methicillin-resistant Staphylococcus aureus.

Like obesity, prolonged food deprivation induces severe hepatic steatosis; however, the functional significance of this phenomenon is not well understood. In this study, we show that the fall in plasma leptin concentration during fasting is required for the development of hepatic steatosis in mice. Removal of leptin receptors from AGRP neurons diminishes fasting-induced hepatic steatosis. Furthermore, the suppressive effects of leptin on fasting-induced hepatic steatosis are absent in mice lacking the gene encoding agouti-related protein (Agrp), suggesting that this function of leptin is mediated by AGRP. Prolonged fasting leads to suppression of hepatic sympathetic activity, increased expression of acyl CoA:diacylglycerol acyltransferase-2 in the liver, and elevation of hepatic triglyceride content and all of these effects are blunted in the absence of AGRP. AGRP deficiency, despite having no effects on feeding or body adiposity in the free-fed state, impairs triglyceride and ketone body release from the liver during prolonged fasting. Furthermore, reducing CNS Agrp expression in wild-type mice by RNAi protected against the development of hepatic steatosis not only during starvation, but also in response to consumption of a high-fat diet. These findings identify the leptin-AGRP circuit as a critical modulator of hepatic triglyceride stores in starvation and suggest a vital role for this circuit in sustaining the supply of energy from the liver to extrahepatic tissues during periods of prolonged food deprivation.

Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-(14)C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.

Human diets contain sterol oxidation products that can induce cytotoxic effects, mainly caused by cholesterol oxides. However, phytosterol oxides effects have been less extensively investigated. This study evaluates the production of inflammatory biomarkers (IL-1β, IL-8, IL-10, TNFα) and the influence of gene expression transporters and enzymes related to cholesterol absorption and metabolism (NPC1L1, ABCG5/8, HMGCoA, ACAT) produced by 7-ketosterols (stigmasterol/cholesterol) in Caco-2 cells. These effects were linked to intracellular signaling pathways by using several inhibitors. Results showed 7-ketostigmasterol to have a greater proinflammatory potential than 7-ketocholesterol. In non-pre-treated cells, only efflux transporters were down-regulated by 7-ketosterols, showing a greater influence upon ABCG5 expression. Cell-pre-incubation with bradykinin induced changes in ABCG expression levels after 7-ketostigmasterol-incubation; however, the energetic metabolism inhibition reduced NPC1L1 expression only in 7-ketocholesterol-incubated cells. In non-pre-treated cells, HMG-CoA was up-regulated by both 7-ketosterols. However, exposure to inhibitors down-regulated the expression levels, mainly in 7-ketocholesterol-incubated cells. While ACAT expression values in non-pre-treated cells were unchanged, exposure to inhibitors caused down-regulation of mRNA levels. These results suggest that internalization and excretion of 7-ketostigmasterol is probably influenced by [Ca]i, which also could mediate HMGCoA activity in POPs metabolism. However, energetic metabolism and reducing equivalents exert different influences upon the 7-ketosterol internalization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ΔphaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction.

Azotobacter vinelandii is a bacterium which undergoes a differentiation process leading to the formation of metabolically dormant cysts. During the encystment process, A. vinelandii produces alkylresorcinol lipids (ARs) that replace the membrane phospholipids and are also components of the layers covering the cyst. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded by the arsABCD operon, which is expressed only during the differentiation process. Also, the production of ARs has been shown to be dependent on the stationary-phase sigma factor RpoS, which is also implicated in the control of the synthesis of other cyst components (i.e., alginate and poly-β-hydroxybutyrate). In this study, we identified ArpR, a LysR-type transcriptional regulator expressed only during encystment that positively regulates arsABCD transcription. We show that this activation is dependent on acetoacetyl-coenzyme A (acetoacetyl-CoA), which might provide a metabolic signal for encystment. We also show that RpoS regulates arsABCD expression through the control of arpR transcription.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the rate-limiting step in the mevalonate pathway. To elucidate the functions of HMGR in triterpene biosynthesis, Platycodon grandiflorum was transformed with a construct expressing Panax ginseng HMGR (PgHMGR). We used PCR analysis to select transformed hairy root lines and selected six lines for further investigation. Quantitative real-time PCR showed higher expression levels of HMGR and total platycoside levels (1.5-2.5-fold increase) in transgenic lines than in controls. Phytosterols levels were also 1.1-1.6-fold higher in transgenic lines than in controls. Among these lines, line T7 produced the highest level of total platycosides (1.60 ± 0.2 mg g(-1) dry weight) and α-spinasterol (1.78 ± 0.16 mg g(-1) dry weight). These results suggest that metabolic engineering of P. grandiflorum by Agrobacterium-mediated genetic transformation may enhance production of phytosterols and triterpenoids.

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying β-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (Km of 0.0029 mM and kcat of 30.0 s(-1)) with no allosteric regulation. Activity toward GAP was lower (Km of 4.6 mM and kcat of 4.77 s(-1)), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.

The endoplasmic reticulum (ER)-resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We interrogated this network in gp78(-/-) mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNA interference. Although we consistently observe involvement of gp78 in Insig-1 degradation, we find no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.

Phoslactomycins (PLMs) are inhibitors of protein serine/threonine phosphatase 2A showing diverse and important antifungal, antibacterial and antitumor activity. PLMs are polyketide natural products and produced by several Streptomyces species. The PLMs biosynthetic gene clusters were identified from Streptomyces platensis SAM-0654 and localized in two separate genomic regions, consisting of 27 open reading frames that encode polyketide synthases (PKSs), enzymes for cyclohexanecarboxyl-CoA (CHC-CoA) and ethylmalonyl-CoA (Em-CoA) synthesis, enzymes for post-PKS modifications, proposed regulators, and putative resistance transporters. Bioinformatic analysis and inactivation experiment of regulatory genes suggest that PnR1 and PnR2 are two positive regulators of PLMs biosynthesis. Gene transcription analysis by reverse transcriptase PCR (RT-PCR) of the PLMs gene cluster demonstrated that PnR1 and PnR2 activate the transcription of the structural biosynthetic genes while PnR2 specially governs the transcription of pnR1 in a higher level., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The antihyperlipidaemic effects of ODP-Ia, one of the main components of Opuntia dillenii Haw. polysaccharides, were studied. Gavage administration of ODP-Ia was observed to significantly decrease serum lipid levels and to increase serum high-density lipoprotein cholesterol level in hyperlipidaemic rats. Similar suppressive patterns were also seen in hepatic total cholesterol and triglyceride levels. Moreover, the ODP-Ia administration significantly increased serum lecithin:cholesterol acyltransferase activity, increased the production of serum NO, inhibited hepatic HMG-CoA reductase activity, augmented serum and hepatic superoxide dismutase activities and decreased the serum and hepatic malondialdehyde contents in hyperlipidaemic rats. In addition, a histopathological examination revealed that ODP-Ia administration significantly suppressed inflammatory cell infiltration and the expression of VCAM-1. Together, these results indicate that ODP-Ia is a potential natural product for the treatment of hyperlipidaemia-related diseases by improving antioxidant levels, modulating the activities of enzymes involved in cholesterol metabolism, promoting the production of NO and suppressing the expression of VCAM-1, thereby suppressing lipid accumulation and inflammatory cell infiltration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening., Methods: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes., Results: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing., Conclusions: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.

DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.

The mucus layer in the colon, acting as a barrier to prevent invasion of pathogens, is thinner and discontinuous in patients with ulcerative colitis (UC). A recent developed in vitro dynamic gut model, the M-SHIME, was used to compare long-term colonization of the mucin layer by the microbiota from six healthy volunteers (HV) and six UC patients and thus distinguish the mucin adhered from the luminal microbiota. Although under the same nutritional conditions, short-chain fatty acid production by the luminal communities from UC patients showed a tendency toward a lower butyrate production. A more in-depth community analysis of those microbial groups known to produce butyrate revealed that the diversity of the Clostridium coccoides/Eubacterium rectale and Clostridium leptum group, and counts of Faecalibacterium prausnitzii were lower in the luminal fractions of the UC samples. Counts of Roseburia spp. were lower in the mucosal fractions of the UC samples. qPCR analysis for butyryl-CoA:acetate CoA transferase, responsible for butyrate production, displayed a lower abundance in both the luminal and mucosal fractions of the UC samples. The M-SHIME model revealed depletion in butyrate producing microbial communities not restricted to the luminal but also in the mucosal samples from UC patients compared to HV., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann-Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Fungal secondary metabolites are a rich source of medically useful compounds due to their pharmaceutical and toxic properties. Sequencing of fungal genomes has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in normal laboratory conditions. Therefore, to discover new metabolites, it is important to find ways to induce the expression of genes in these otherwise silent biosynthetic clusters. We discovered a novel secondary metabolite in Aspergillus nidulans by predicting a biosynthetic gene cluster with genomic mining. A Zn(II)(2)Cys(6)-type transcription factor, PbcR, was identified, and its role as a pathway-specific activator for the predicted gene cluster was demonstrated. Overexpression of pbcR upregulated the transcription of seven genes in the identified cluster and led to the production of a diterpene compound, which was characterized with GC/MS as ent-pimara-8(14),15-diene. A change in morphology was also observed in the strains overexpressing pbcR. The activation of a cryptic gene cluster by overexpression of its putative Zn(II)(2)Cys(6)-type transcription factor led to discovery of a novel secondary metabolite in Aspergillus nidulans. Quantitative real-time PCR and DNA array analysis allowed us to predict the borders of the biosynthetic gene cluster. Furthermore, we identified a novel fungal pimaradiene cyclase gene as well as genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a geranylgeranyl pyrophosphate (GGPP) synthase. None of these genes have been previously implicated in the biosynthesis of terpenes in Aspergillus nidulans. These results identify the first Aspergillus nidulans diterpene gene cluster and suggest a biosynthetic pathway for ent-pimara-8(14),15-diene.

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 Å of each other provides the basis for enhanced in vivo synthesis of resveratrol., (© 2011 American Chemical Society)

Background: Succinyl-coenzyme A ligase (SUCL) is a mitochondrial enzyme that catalyses the reversible conversion of succinyl-coenzyme A to succinate. SUCL consists of an α subunit, encoded by SUCLG1, and a β subunit, encoded by either SUCLA2 or SUCLG2. Recently, mutations in SUCLG1 or SUCLA2 have been identified in patients with infantile lactic acidosis showing elevated urinary excretion of methylmalonate, mitochondrial respiratory chain (MRC) deficiency, and mitochondrial DNA depletion., Methods: Case description of a Japanese female patient who manifested a neonatal-onset lactic acidosis with urinary excretion of methylmalonic acid. Enzymatic analyses (MRC enzyme assay and Western blotting) and direct sequencing analysis of SUCLA2 and SUCLG1 were performed., Results: MRC enzyme assay and Western blotting showed that MRC complex I was deficient. SUCLG1 mutation analysis showed that the patient was a compound heterozygote for disease-causing mutations (p.M14T and p.S200F)., Conclusion: For patients showing neonatal lactic acidosis and prolonged mild methylmalonic aciduria, MRC activities and mutations of SUCLG1 or SUCLA2 should be screened for., (© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.)

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the V(max) of the enzyme for glycerolipids but had only a modest effect on the K(m). Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities.

We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C(16) and C(18) fatty acids into TAG., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.

The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza., (Copyright © 2010 Elsevier GmbH. All rights reserved.)