Dual sites of occurrence of malonyl-CoA decarboxylase and their possible functional significance in avian tissues.

1. Uropygial glands of domestic goose and mallard which synthesize methyl-branched fatty acids, contain large quantities of cytosolic malonyl-CoA decarboxylase and a small quantity of mitochondrial enzyme. 2. Uropygial glands of chicken and the liver of geese which generate little methyl-branched acids, contain only small quantities of malonyl-CoA decarboxylase and in such cases the enzyme is in the mitochondria. 3. The mitochondrial decarboxylase from the uropygial gland and liver of goose is immunologically similar to the cytosolic decarboxylase of the uropygial gland. 4. The mitochondrial enzyme probably protects the mitochondrial enzymes which are susceptible to inhibition by malonyl-CoA, whereas the cytosolic enzyme promotes the synthesis of methyl-branched acids.     

Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function

Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) $\text{Arch. Biochem. Biophys.}$ 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [³⁵S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme.     

Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.     

Malonyl-CoA decarboxylase from the uropygial gland of waterfowl: purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched fatty acids.

Analysis of the acyl portion of the wax from the uropygial gland of muscovy duck, wood duck, (Cairininae subfamily) and Canadian goose (Anserinae) by combined gas-liquid chromatography and mass spectrometry showed that 2,4,6-trimethyloctanoic acid and 2,4,6-trimethylnonanoic acid were the major (~100%) components. Similar analyses of the wax from the glands of mallard and Peking duck (Anatinae) showed that 2- and 4-mono-methylhexanoic acids predominated (>75%) with no multimethyl-branched acids. The uropygial glands of the former group contained 20 to 100 times as much malonyl-CoA decarboxylase activity as those of the latter group. These results strongly support the hypothesis that this decarboxylase, by causing specific decarboxylation of malonyl-CoA, makes available only methylmalonyl-CoA for fatty acid synthesis, and thus causes the production of multimethyl-branched acids. Malonyl-CoA decarboxylase was purified to apparent homogeniety in 30% yield from the uropygial glands of muscovy and wood ducks. Properties of the enzyme from the ducks, such as S_20.w (7.8 S), molecular weight (190,000) subunit composition (4 × 47,000), amino acid composition, strict substrate specificity, pH optimum (~9.0), K_m (~33 μm), V (~80 μmol/min/mg), and inhibition by SH-directed reagents were similar to those observed with the decarboxylase from the domestic goose. Antiserum prepared against the goose enzyme cross-reacted with and inhibited the decarboxylase from the four genera of ducks and Canadian goose. Ouchterlony double-diffusion analyses showed fusion of precipitant lines with the enzyme from muscovy, wood duck, and Canadian goose, whereas spurs were observed with the enzymes from mallard and Peking ducks. Immunoelectrophoresis showed that the decarboxylases from muscovy and wood ducks were similar and that they were different from the enzyme from the domestic goose. It appears that during evolution, the subfamilies (Anserinae and Cairininae) which synthesize multimethyl-branched acids acquired the ability to produce a high level of malonyl-CoA decarboxylase, an enzyme which is also present in low levels in other organisms.     

Lipid biosynthesis in sebaceous glands: regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase.

Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.     

Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression

Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.     

Stereospecificity of malonyl-CoA decarboxylase, acetyl-CoA carboxylase, and fatty acid synthetase from the uropygial gland of goose

Malonyl-CoA decarboxylase from the uropygial gland of goose decarboxylated (R,S)-methylmalonyl-CoA at a slow rate and introduced 3H from [3H]2O into the resulting propionyl-CoA. Carboxylation of this labeled propionyl-CoA by propionyl-CoA carboxylase from pig heart and acetyl-CoA carboxylase from the uropygial gland completely removed 3H. Repeated treatment of (R,S)-[methyl-14C]methylmalonyl-CoA with the decarboxylase converted 50% of the substrate into propionyl-CoA, whereas (S)-methylmalonyl-CoA, generated by both carboxylases, was completely decarboxylated. Radioactive (R)- (S), and (R,S)-methylmalonyl-CoA were equally incorporated into fatty acids by fatty acid synthetase from the uropygial gland. The residual methylmalonyl-CoA remaining after fatty acid synthetase reaction on (R,S)-methylmalonyl-CoA was also racemic. These results show that: (a) the decarboxylase is stereospecific, (b) replacement of the carboxyl group by hydrogen occurs with retention of configuration, (c) acetyl-CoA carboxylase of the uropygial gland generates (S)-methylmalonyl-CoA from propionyl-CoA, and (d) fatty acid synthetase is not stereospecific for methylmalonyl-CoA.     

Lipid biosynthesis in the sebaceous glands: synthesis of multibranched fatty acids from methylmalonyl-coenzyme A in cell-free preparations from the uropygial gland of goose.

Cell-free extracts from the uropygial gland of goose catalyzed the incorporation of malonyl-CoA and methylmalonyl-CoA into n- and multi-branched fatty acids, respectively, with NADPH as the preferred reductant. Methylmalonyl-CoA was shown to be incorporated almost exclusively into the acyl portion of wax esters by the cell-free extract while malonyl-CoA was incorporated into polar lipids and both the acyl and alcohol portions of the wax. The optimal pH for the synthesis of both n- and multibranched acids was 6.0. Apparent Km and Vmax for malonyl-CoA were 2 times 10- minus-4 M and 250 nmol per min per mg, respectively, while the Km and Vmax for methylmalonyl-CoA were 7.7 times 10- minus-4 M and 0.8 nmol per min per mg, respectively with 105,000g supernatant; but partial purification resulted in a tenfold decrease in Km values. The partially purified synthetase preparation catalyzed the formation of n-C16 acid (80%) and n-C18 acid (20%) from acetyl-CoA and malonyl-CoA. With the same synthetase preparation and the appropriate primer methylmalonyl-CoA was converted into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid which were identified by radio gas-liquid chromatography and combined gas chromatography-mass spectrometry. Experiments with an equimolecular mixture of acetyl-CoA and propionyl-CoA showed that the synthetase preferred acetyl-CoA as a primer. Since malonyl-CoA is known to be rapidly decarboxylated in the gland, acetyl-CoA and methylmalonyl-CoA are expected to be the major primer and elongating agent, respectively, available in the gland and therefore 2,4,6,8-tetramethyldecanoic acid should be the major product. Combined gas-liquid chromatography and mass spectrometry demonstrated that this acid was in fact the major acid of the gland.     

Purification and properties of malonyl-CoA decarboxylase from rat liver mitochondria and its immunological comparison with the enzymes from rat brain, heart, and mammary gland.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH₄)₂SO₄ precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which K_m and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.     

Primary structure of a chymotryptic peptide containing the "active serine" of the thioesterase domain of fatty acid synthase

“Active serine” of the thioesterase domain of fatty acid synthase from the goose uropygial gland was selectively labeled with [1,3-³H]diisopropylfluorophosphate and the chymotryptic peptide containing this active serine was purified to homogeneity by a combination of gel filtration, cation exchange chromatography and high performance liquid chromatography. The primary structure of this active site peptide, Ser-Phe-Gly-Ala-Cys-Val-Ala-Phe, is remarkably homologous to the “active serine” containing peptide of human plasmin.     

MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO(2). Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucine(COOH). We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947-948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal beta-oxidation of odd chain-length dicarboxylic fatty acids.     

Evidence for an "active serine" in each fatty acid synthetase peptide.

Ultracentrifugally homogeneous fatty acid synthetase was isolated from the uropygial gland of goose by a one-step purification procedure. Formation of fatty acids from malonyl-CoA and hydrolysis of palmitoyl-CoA catalyzed by the synthetase were inhibited to an equal extent by diisopropylfluorophosphate. With labeled inhibitor, it was shown that one mole of the inhibitor was covalently attached per mole of the subunit of the enzyme. Sodium dodecyl sulphate electrophoresis showed that all of the label was contained in a 270,000 M.Wt peptide. That the active serine was not at the loading site was suggested by the observations that neither acetylation nor malonylation of the enzyme affected the reaction of the enzyme with the inhibitor and acetylation or malonylation of the enzyme was not affected by this inhibitor. Thus, each fatty acid synthetase peptide is shown to have one active serine which most probably is at the chain terminating active site of the peptide.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.     

Coupling of the biosynthesis of fatty acids and fatty alcohols.

A cell-free system for the biosynthesis of fatty alcohols in the pink portion of the rabbit harderian gland is described. The radiolabeled substrates for the fatty acid reductase were generated using soluble fatty acid synthase from the gland in the presence of acetyl-CoA, malonyl-CoA, and NADPH. Harderian gland microsomes, ATP, and Mg²⁺ were absolute requirements for the synthesis of fatty alcohols and if any of these components were deleted from the assay mixture, no alcohols were detected. We were also unable to detect formation of fatty alcohols if acyl-CoAs were substituted for fatty acid synthase with either NADPH or NADH as reducing agents. The reductase was localized in the microsomal fraction and appears to be on the cytosol-membrane interface of the vesicles, as indicated in experiments using detergents and trypsin. The fatty alcohols formed by the system had the same chain length distribution as the fatty acids made by the fatty acid synthase. The alkyl moieties of the ether lipids in the harderian gland are exclusively saturated and the properties of the alcohol-synthesizing system described in this report can account for the observed exclusion of unsaturated alkyl moieties from the ether lipids of this gland.