Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.

Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.

Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 μmol acetoacetate formed min−1⋅mg−1 at 30°C and pH 7.6 and apparent Km values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

The vitamin K-dependent γ-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to γ-carboxyglutamic acid in precursor proteins containing the γ-carboxylation recognition site (γ-CRS). During this reaction, glutamic acid is converted to γ-carboxyglutamic acid while vitamin KH2 is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of γ-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We show that in the absence of γ-CRS-containing propeptide and/or glutamate-containing substrate, carboxylase has little or no epoxidase activity. Epoxidase activity is induced by Phe-Leu-Glu-Glu-Leu (FLEEL) (9.2 pmol per min per pmol of enzyme), propeptide, residues −18 to −1 of proFactor IX (3.4 pmol per min per pmol of enzyme), FLEEL and propeptide (100 pmol per min per pmol of enzyme), and proPT28 (HVFLAPQQARSLLQRVRRANTFLEEVRK, residues −18 to +10 of human acarboxy-proprothrombin), (5.3 pmol per min per pmol of enzyme). These results indicate that in the absence of propeptide or glutamate-containing substrate, oxygenation of vitamin K by the carboxylase does not occur. Upon addition of propeptide or glutamate-containing substrate, the enzyme is converted to an active epoxidase. This regulatory mechanism prevents the generation of a highly reactive vitamin K intermediate in the absence of a substrate for carboxylation.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II−. Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by ≈6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of ≈0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II− requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.

Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Immunophilins, Refsum disease, and lupus nephritis: The peroxisomal enzyme phytanoyl-COA α-hydroxylase is a new FKBP-associated protein

FKBP52 (FKBP59, FKBP4) is a “macro” immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12. To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role. This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein. Determination of the sequence of this protein permitted its identification as phytanoyl-CoA α-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein. Inactivation of this enzyme is responsible for Refsum disease in humans. The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis. We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52. Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506. This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO2, generating the γ-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684–1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH2 preincubation. Amino acid analysis of 14C- N-ethyl maleimide-modified human carboxylase revealed 1.8–2.3 reactive residues and a specific activity of 7 × 108 cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the Kms for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH2 oxygenation.

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: The phenylacetyl-CoA catabolon

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a β-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 β-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.