Malonyl CoA control of fatty acid oxidation in the newborn heart in response to increased fatty acid supply

The concentration of fatty acids in the blood or perfusate is a major determinant of the extent of myocardial fatty acid oxidation. Increasing fatty acid supply in adult rat increases myocardial fatty acid oxidation. Plasma levels of fatty acids increase post-surgery in infants undergoing cardiac bypass operation to correct congenital heart defects. How a newborn heart responds to increased fatty acid supply remains to be determined. In this study, we examined whether the tissue levels of malonyl CoA decrease to relieve the inhibition on carnitine palmitoyltransferase (CPT) I when the myocardium is exposed to higher concentrations of long-chain fatty acids in newborn rabbit heart. We then tested the contribution of the enzymes that regulate tissue levels of malonyl CoA, acetyl CoA carboxylase (ACC), and malonyl CoA decarboxylase (MCD). Our results showed that increasing fatty acid supply from 0.4 mmol/L (physiological) to 1.2 mmol/L (pathological) resulted in an increase in cardiac fatty acid oxidation rates and this was accompanied by a decrease in tissue malonyl CoA levels. The decrease in malonyl CoA was not related to any alterations in total and phosphorylated acetyl CoA carboxylase protein or the activities of acetyl CoA carboxylase and malonyl CoA decarboxylase. Our results suggest that the regulatory role of malonyl CoA remained when the hearts were exposed to high levels of fatty acids.     

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.     

Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.     

The Bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl‐CoA carboxylase

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate‐limiting step in fatty acid biosynthesis is catalyzed by a multi‐subunit form of the acetyl‐CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl‐CoA to produce malonyl‐CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the P_II family. Here we show that the GlnB paralogues of the P_II proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC‐BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC‐BCCP‐GlnB interaction and ACC inhibition were relieved by 2‐oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2‐oxoglutarate‐sensitive dissociable regulatory subunit of ACC in Bacteria.     

Rapid Purification of Enzymes of Fatty Acid Biosynthesis from Rat Adipose Tissue

Abstract

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protien kinases.     

Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism

The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.     

地中海拟无枝菌酸菌U32中生物素羧基载体蛋白结构基因的克隆、表达及转录

乙酰辅酶A羧化酶(Acetyl CoA Carboxylase EC 6.4.1.2, ACC)催化依赖于ATP的乙酰辅酶A羧化形成丙二酸单酰辅酶A,该反应是脂肪酸生物合成途径中的第一步,也是受到调控的关键一步。根据结核分枝杆菌(M. tuberculosis)和天蓝色链霉菌(S. coelicolor)中ACC-α亚基的氨基酸保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好,设计简并引物以U32基因组DNA为模板扩增出一条约250bp的片段,并以此片段作探针成功地从U32基因组cosmid文库中克隆到相应的ACC-α亚基的编码基因accA。该基因对应的ORF长1797bp,编码一个598个氨基酸的蛋白,推算出的分子量是63,714Da;基因G+C mol%含量为70.1%,符合U32基因结构特征,距起始密码子GTG上游6个碱基处有链霉菌典型的RBS序列AGGAGG,并有生物素羧化酶特征的ATP结合区。利用pET28(b)系统构建表达载体,在E. coli BL21(DE3)中实现了accA的诱导表达,产物大部分以可溶形式存在,并通过Western Blot证明该蛋白上确有共价结合的生物素。Northern Blot分析了各种氮源对accA基因转录水平的不同影响。     

Effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase

The effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase has been investigated. Citrate showed modest effect on the ATP-orthophosphate and ATP-ADP exchange reactions. In contrast, this tricarboxylic acid caused marked concentration-dependent stimulation of the acetyl CoA-malonyl CoA exchange reaction which was concomitant with the activation of acetyl CoA carboxylation. The data obtained are consistent with the suggestion that activation by citrate of the overall forward reaction (malonyl CoA synthesis) primarily reflects enhancement of the carboxyltransferase half-reaction catalyzed by rat mammary gland acetyl CoA carboxylase.     

Cyclohexanedione herbicides are inhibitors of rat heart acetyl-CoA carboxylase

Acetyl CoA carboxylase (ACC) catalyzes the carboxylation of acetyl CoA to form malonyl CoA. In skeletal muscle and heart, malonyl CoA functions to regulate lipid oxidation by inhibition of carnitine palmitoyltransferase-1, an enzyme which controls the entry of long chain fatty acids into mitochondria. We have found that several members of the cyclohexanedione class of herbicides are competitive inhibitors of rat heart ACC. These compounds constitute valuable reagents for drug development and the study of ACCbeta, a validated anti-obesity target.     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

De novo synthesis and elongation of fatty acids by subcellar fractions of monkey aorta

Subcellular fractions of aorta of squirrel monkey (Saimiri sciureus) were examined for their ability to synthesize and elongate fatty acids. High-speed supernate (HSS) incorporated substantial quantities of malonyl CoA into fatty acids while acetyl CoA was much less effectively utilized. Acetyl-CoA carboxylase activity exceeded the amount of acetyl CoA incorporated into fatty acids and thus does not account for the low incorporation of this substrate. Microsomes used malonyl CoA and acetyl CoA equally well; mitochondria incorporated either acetyl CoA or acetate. The amounts of substrate incorporated into fatty acids (m micro moles/mg of protein per hr) were 2.3 for HSS, 1.2 for microsomes, and 0.9 for mitochondria. The synthesized fatty acids were separated by gas-liquid chromatography, radioassayed, extracted from the scintillation fluid, and decarboxylated. HSS completely synthesized palmitic and stearic acids from malonyl CoA. Microsomes and mitochondria utilized acetyl CoA to elongate endogenous fatty acids and gave mainly palmitic, stearic, and C(18) and C(20) monoenoic acids, with lesser amounts of other saturated and unsaturated fatty acids. A significant quantity of malonyl CoA was utilized by microsomes to yield a fatty acid tentatively identified as docosapentaenoic. Radioactive fatty acids are incorporated into various lipid classes by the particulate preparations. These studies demonstrate that aortic tissue in a nonhuman primate is able to carry out several processes of fatty acid metabolism and that the aortic synthesis and elongation of fatty acids may play an important role in providing fatty acids for incorporation into aortic lipids.     

Irreversible inactivation of chicken liver fatty acid synthetase by its substrates acetyl and malonyl CoA: Effect of temperature and NADP+ on fatty acid and triacetic acid lactone synthesis

Chicken liver fatty acid synthetase is irreversibly inactivated by malonyl CoA and by acetyl and malonyl CoA. Two active forms of the enzyme existing above and below 11.5° are inactivated at different rates. Activities for fatty acid and triacetic acid lactone synthesis are lost at about the same rate and NADP⁺ protects the enzyme against inactivation. Inactivation results from the enhanced covalent binding of malonyl groups in addition to those required for fatty acid synthesis.     

Inhibitory effect of acetazolamide on the activity of acetyl CoA carboxylase of mouse liver.

The effect of different concentrations of acetazolamide on the activities of acetyl CoA carboxylase and fatty acid synthetase was studied. Acetazolamide inhibits the activity of 100, 00 × g supernatant acetyl CoA carboxylase and that of this purified enzyme even in the presence of high concentrating of bicarbonate. It is without action on fatty acid synthetasse.     

Site-directed mutagenesis of ATP binding residues of biotin carboxylase. Insight into the mechanism of catalysis

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N(5)-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K(m) value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)     

Malonyl-CoA and AMP-activated protein kinase: An expanding partnership

Insulin resistance in skeletal muscle is present in humans with type 2 diabetes (noninsulin-dependent diabetes mellitus) and obesity and in rodents with these disorders. Malonyl CoA is a regulator of carnitine palmitoyl transferase I (CPT I), the enzyme that controls the transfer of long chain fatty acyl CoA into mitochondria where it is oxidized. In rat skeletal muscle, the formation of malonyl CoA is regulated acutely (in minutes) by changes in the activity of acetyl CoA carboxylase (ACC), the enzyme that catalyzes malonyl CoA synthesis. Acc activity can be regulated by changes in the concentration of citrate which is both an allosteric activator of Acc and a source of its precursor, cytosolic acetyl CoA. Increases in cytosolic citrate leading to an increase in the concentration of malonyl CoA occur when muscle is presented with insulin and glucose, or when it is made inactive by denervation. In contrast, exercise lowers the concentration of malonyl CoA, by activating an AMP activated protein kinase (AMPK), which phosphorylates and inhibits ACC. Recently we have shown that the activity of malonyl CoA decarboxylase (MCD), an enzyme that degrades malonyl CoA, is also regulated by phosphorylation. The concentration of malonyl CoA in liver and muscle in certain circumstances correlates inversely with changes in MCD activity. This review will describe the current literature on the regulation of malonyl CoA/AMPK mechanism and its physiological function.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.