Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

Late diagnosis of primary hyperoxaluria type III.

We report the case of a 78-year-old patient with late diagnosis of hyperoxaluria type III (PH3). He developed renal failure after nephrectomy for clear cell papillary renal carcinoma and complained of recurrent urolithiasis for some 30 years, whose aetiology was never identified. Biochemical laboratory investigations of urine and urolithiasis composition revealed marked hyperoxaluria but normal concentrations of urinary glyceric and glycolic acid as well as stones of idiopathic calcium-oxalate appearance. Furthermore, the dietary survey showed excessive consumption of food supplements containing massive amounts of oxalate precursors. However, the persistence of excessive hyperoxaluria after his eating habits was changed leading us to perform molecular genetic testing. We found heterozygous mutations of the recently PH3-associated HOGA1 gene when sequencing PH genes. This is the first description of late diagnosis primary PH3 in a patient with several additional pro-lithogenic factors. This case illustrates the importance of undertaking a complete biological work-up to determine the aetiology of hyperoxaluria. This may reveal underdiagnosed primary hyperoxaluria, even in older patients.

Pharmacological inhibition of FTO.

In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.

Cut-like homeobox 1 (CUX1) regulates expression of the fat mass and obesity-associated and retinitis pigmentosa GTPase regulator-interacting protein-1-like (RPGRIP1L) genes and coordinates leptin receptor signaling.

The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.

Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase.

Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of approximately 41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases, and enzymes involved in lipid metabolism.

Evaluation of lysine biosynthesis as an antifungal drug target: biochemical characterization of Aspergillus fumigatus homocitrate synthase and virulence studies.

Aspergillus fumigatus is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal alpha-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence. A. fumigatus HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and alpha-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An hcsA deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified A. fumigatus proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation.

Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

Hypothalamic FTO is associated with the regulation of energy intake not feeding reward.

BACKGROUND: Polymorphism in the FTO gene is strongly associated with obesity, but little is known about the molecular bases of this relationship. We investigated whether hypothalamic FTO is involved in energy-dependent overconsumption of food. We determined FTO mRNA levels in rodent models of short- and long-term intake of palatable fat or sugar, deprivation, diet-induced increase in body weight, baseline preference for fat versus sugar as well as in same-weight animals differing in the inherent propensity to eat calories especially upon availability of diverse diets, using quantitative PCR. FTO gene expression was also studied in organotypic hypothalamic cultures treated with anorexigenic amino acid, leucine. In situ hybridization (ISH) was utilized to study FTO signal in reward- and hunger-related sites, colocalization with anorexigenic oxytocin, and c-Fos immunoreactivity in FTO cells at initiation and termination of a meal. RESULTS: Deprivation upregulated FTO mRNA, while leucine downregulated it. Consumption of palatable diets or macronutrient preference did not affect FTO expression. However, the propensity to ingest more energy without an effect on body weight was associated with lower FTO mRNA levels. We found that 4-fold higher number of FTO cells displayed c-Fos at meal termination as compared to initiation in the paraventricular and arcuate nuclei of re-fed mice. Moreover, ISH showed that FTO is present mainly in hunger-related sites and it shows a high degree of colocalization with anorexigenic oxytocin. CONCLUSION: We conclude that FTO mRNA is present mainly in sites related to hunger/satiation control; changes in hypothalamic FTO expression are associated with cues related to energy intake rather than feeding reward. In line with that, neurons involved in feeding termination express FTO. Interestingly, baseline FTO expression appears linked not only with energy intake but also energy metabolism.

Assessing the metabolic and toxic effects of anticonvulsant doses of polyunsaturated fatty acids on the liver in rats.

Polyunsaturated fatty acids (PUFA), at high doses, have been demonstrated to possess anticonvulsant properties in animal seizure models. Little is known, however, about the possible metabolic or adverse effects of PUFA at these high, anticonvulsant doses. The goal of the present study was to assess the metabolic and potential adverse effects of high-dose PUFA administration to rats. Adult male rats received a fatty acid mixture containing alpha-linolenic and linoleic acid in a 1 to 4 ratio, intraperitoneally, for 3 wk. After sacrifice, livers were isolated and analyzed for fatty acid composition and for mRNA expression of HMG-CoA lyase, catalase, and glutathione S-transferases A1 and A4, markers for ketosis, antioxidant defense, and phase II xenobiotic metabolism, respectively. Chronic administration of the PUFA mixture decreased hepatic levels of total lipids--and several fatty acids within total lipids--without altering mRNA expression of HMG-CoA lyase, a metabolic marker of ketosis. The PUFA mixture did not affect mRNA expression of catalase or glutathione S-transferases A1 and A4, which are involved in antioxidant defense and phase II xenobiotic metabolism. These findings suggest that PUFA, given for 3 wk at anticonvulsant doses, result in significant changes in liver lipid metabolism, but do not alter measured genetic markers of liver toxicity.

Regulation of Fto/Ftm gene expression in mice and humans.

Two recent, large whole-genome association studies (GWAS) in European populations have associated a approximately 47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4 degrees C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lep ob, Lepr db, Cpe fat, or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 preferentially bound CUTL1[corrected] in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.

The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase.

In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

Production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) using enzymes or whole cells entrapped in calcium pectate-silica-gel beads.

The present study focuses on the application of immobilization technology to enzymic sugar syntheses. The paper describes an improved silica-alginate matrix established for entrapment and encapsulation. The replacement of alginate with pectate provided enhanced chemical resistance of the matrix, which allows the use of 1% (w/v) polyphosphate in reaction mixtures. Polylysine, a reagent for silica condensation, was replaced by a much cheaper alternative, namely polyethyleneimine. The proposed design was applied in the production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) by immobilized recombinant enzymes or Escherichia coli cells containing overexpressed enzymes. A comparison between these two strategies was made. On the basis of the results we conceptualized a system to synthesize sialyloligosaccharides by using a biocatalyst entrapped in calcium pectate-silica gel beads.

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate.

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.

Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture.

Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.

Purification and characterization of glyoxylate synthetase from greening potato-tuber chloroplasts.

Glyoxylate synthetase catalyzing the condensation of two formate molecules into glyoxylate was purified to homogeneity by AcA-34, Sepharose CL-6B and DEAE-Sepharose CL-6B chromatography. A 150-fold purification with a specific activity of 25 mumol . mg protein-1 x 5 min-1 was obtained by this procedure. The reaction product was identified as glyoxylate. The enzyme was a tetramer having a molecular mass of 160 kDa with a subunit molecular mass of 40 kDa. The enzyme could be activated 3-4-fold by the addition of 0.3 mM Fe2+ and 0.4 mM tetrahydrofolic acid to the reaction mixture. The requirement for Fe2+ and tetrahydrofolic acid was confirmed from the inhibition of enzyme by O-phenanthroline and alpha-aminopterin, respectively. The presence of a bound folate in the enzyme was indicated by the fluorescence emission at 450 nm and turbidity development in a Lactobacillus casei growth test. Fluorescence emission at 450 nm upon excitation at 280 nm indicated that the bound folate and the aromatic amino-acid residues of the enzyme were in close vicinity. The enzyme was maximally active at 25 degrees C and exhibited a pH optimum at 7.0. The concentration of substrate was optimal at 5.0 mM and Km for substrate was found to be 1.4 mM. Activation by Fe2+ did not alter the Km but caused an increase in Vmax. The enzyme contained about 14-16 disulfide linkages, of which two were found to be reduced by treatment with 2-mercaptoethanol. The presence of excess 2-mercaptoethanol in the enzyme was inhibitory, indicating that the two disulfide linkages reduced by 2-mercaptoethanol were essential for activity. This was also confirmed by the inhibition of enzyme activity when reduced enzyme was treated with O-phthalaldehyde, which formed a thioisoindole derivative with reduced thiol groups at the active site.

Oxalate metabolism in magnesium-deficient rats.

Male weanling rats were maintained on magnesium-deficient diet for 30 d and compared with pair-fed control rats fed magnesium-supplemented diet. Magnesium deficiency led to slow growth and finally to a significant decrease in body weight (P < 0.001) accompanied by a significant hypomagnesaemia, hypomagnesuria and hyperoxaluria (P < 0.001 in each case) in experimental rats as compared to the control rats. Magnesium deficiency altered the glyoxylate metabolism in the liver and kidney mitochondria by significantly decreasing glyoxylate oxidation (by 26 per cent in liver and 17 per cent in kidney) and activity of alpha-ketoglutarate:glyoxylate carboligase enzyme (by 35 per cent in liver and 27 per cent in kidney) in the experimental animals. A significant increase in the specific activities of glycolic acid oxidase (P < 0.001) and glycolic acid dehydrogenase (P < 0.01) and a significant decrease in alanine transaminase (P < 0.01) was also observed in magnesium-deficient rats. No change in liver and kidney lactate dehydrogenase was observed. Thus magnesium deficiency in rats leads to accumulation of glyoxylate in the tissues, a part of which is converted into oxalate, thereby promoting hyperoxaluria.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at -80 degrees C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM). While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of hepatic cholesterol biosynthesis by fatty acids: effect of feeding olive oil on cytoplasmic acetoacetyl-coenzyme A thiolase, beta-hydroxy-beta-methylglutaryl-CoA synthase, and acetoacetyl-coenzyme A ligase.

We reported previously that, in the perfused rat liver, oleic acid increased the specific activity of cytosolic enzymes of cholesterol biosynthesis. In this study, we examined the effects of oral administration of olive oil on the activities of HMG-CoA synthase, AcAc-CoA thiolase, AcAc-CoA ligase and HMG-CoA reductase. Olive oil feeding increased the specific activity of hepatic HMG-CoA synthase by 50%, AcAc-CoA thiolase by 2-fold, and AcAc-CoA ligase by 3-fold. Olive oil had no effect on HMG-CoA reductase activity. These data suggest that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are regulated in parallel by a physiological substrate, fatty acid, independent of HMG-CoA reductase under these conditions.