The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in baker's yeast

Bucovaz, E.T.; MacLeod, Robert M.; Morrison, John C.; Whybrew, W.D.

doi:10.1016/s0300-9084(97)86938-6

Cited by 21 publications

(21 citation statements)

References 12 publications

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“…Using isolated yeast mitochondria, we were unable to apply these findings for setting up a transport assay for CoA. This was mainly due to the fact that radiolabeled CoA was rapidly metabolized when added to isolated yeast mitochondria, presumably by cleavage of CoA to 4-phosphopantetheine, a reaction catalyzed by CoA hydrolase (5). The data presented here fit nicely with the cytosolic synthesis of CoA, as ⌬leu5 cells were capable of producing normal levels of cytosolic CoA.…”

Section: Discussionmentioning

confidence: 80%

The Yeast Mitochondrial Carrier Leu5p and Its Human Homologue Graves' Disease Protein Are Required for Accumulation of Coenzyme A in the Matrix

Prohl

Pelzer

Diekert

et al. 2001

Molecular and Cellular Biology

102

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The transport of metabolites, coenzymes, and ions across the mitochondrial inner membrane is still poorly understood. In most cases, membrane transport is facilitated by the so-called mitochondrial carrier proteins. The yeast Saccharomyces cerevisiae contains 35 members of the carrier family, but a function has been identified for only 13 proteins. Here, we investigated the yeast carrier Leu5p (encoded by the gene YHR002w) and its close human homologue Graves' disease protein. Leu5p is inserted into the mitochondrial inner membrane along the specialized import pathway used by carrier proteins. Deletion of LEU5 (strain ⌬leu5) was accompanied by a 15-fold reduction of mitochondrial coenzyme A (CoA) levels but did not affect the cytosolic CoA content. As a consequence, the activities of several mitochondrial CoA-dependent enzymes were strongly decreased in ⌬leu5 cells. Our in vitro and in vivo analyses assign a function to Leu5p in the accumulation of CoA in mitochondria, presumably by serving as a transporter of CoA or a precursor thereof. Expression of the Graves' disease protein in ⌬leu5 cells can replace the function of Leu5p, demonstrating that the human protein represents the orthologue of yeast Leu5p. The function of the human protein might not be directly linked to the disease, as antisera derived from patients with active Graves' disease do not recognize the protein after expression in yeast, suggesting that it does not represent a major autoantigen. The two carrier proteins characterized herein are the first components for which a role in the subcellular distribution of CoA has been identified.Mitochondria perform a variety of processes, such as oxidative phosphorylation, the citric acid cycle, the ␤-oxidation of fatty acids, parts of the urea cycle, and the biosynthesis of heme and certain amino acids (13,14,61). The metabolic activity of mitochondria requires the rapid and highly specific exchange of molecules between the cytosol and the mitochondrial matrix space. To a large extent, this is facilitated by a family of transport proteins of the inner membrane, the so-called mitochondrial carrier proteins (for reviews, see references 20, 22, 31, 44, 45, and 67). Members of this family include proteins responsible for the exchange of ADP and ATP (termed AAC or ANT) and for the transport of, e.g., phosphate, citrate, carnitine, dicarboxylates, amino acids, flavin adenine dinucleotide (FAD), or protons. The biogenesis of carriers differs in various aspects from that of most other mitochondrial proteins (reviewed in references 2, 32, and 60). They lack an N-terminal targeting sequence (presequence) and they follow a unique import pathway involving the interaction with specialized import components in the outer membrane (Tom70), the intermembrane space (Tim8, Tim9, Tim10, Tim12, and Tim13), and the inner membrane (Tim18, Tim22, and Tim54).In their transport-competent form, carrier proteins are dimeric (50). Each monomer is comprised of three homologous modules containing two transmembrane segments each. Both ...

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Section: Discussionmentioning

confidence: 80%

The Yeast Mitochondrial Carrier Leu5p and Its Human Homologue Graves' Disease Protein Are Required for Accumulation of Coenzyme A in the Matrix

Prohl

Pelzer

Diekert

et al. 2001

Molecular and Cellular Biology

102

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“…This was verified by Worrall and Tubbs, who purified a protein from pork liver that possessed both the phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities (25). Subsequently, these two activities were shown to be part of a multifunctional enzyme complex in baker's yeast (5,6). We first showed that the final two steps in CoA biosynthesis in Corynebacterium ammoniagenes (formerly Brevibacterium ammoniagenes) are catalyzed by distinct proteins that were readily separated by ion-exchange chromatography (12).…”

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confidence: 86%

Identification of yacE ( coaE ) as the Structural Gene for Dephosphocoenzyme A Kinase in Escherichia coli K-12

2001

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Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.Coenzyme A (CoA) is an essential cofactor in a wide variety of biochemical pathways (1). It is estimated that about 4% of all enzymes use CoA or a thioester of CoA as a substrate (24). The final two steps in CoA biosynthesis are coupling of phosphopantetheine with ATP to form dephosphocoenzyme A (dephospho-CoA) and the subsequent phosphorylation of the 3Ј-hydroxyl group to form CoA ( Fig. 1) (3). Genes coding for six of the seven enzymes involved in the biosynthesis of phosphopantetheine have been identified (10,14,20,26). The final two reactions of CoA biosynthesis in mammalian cells were first attributed to a single bifunctional enzyme by Hoagland and Novelli (8). This was verified by Worrall and Tubbs, who purified a protein from pork liver that possessed both the phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities (25). Subsequently, these two activities were shown to be part of a multifunctional enzyme complex in baker's yeast (5, 6). We first showed that the final two steps in CoA biosynthesis in Corynebacterium ammoniagenes (formerly Brevibacterium ammoniagenes) are catalyzed by distinct proteins that were readily separated by ion-exchange chromatography (12). The gene from Escherichia coli coding for phosphopantetheine adenylyltransferase was recently cloned, and the crystal structure of the enzyme was determined (7, 9). We report here purification of dephospho-CoA kinase from wildtype C. ammoniagenes and identification of the gene coding for a homologous protein in E. coli. This gene was cloned using PCR and overexpressed, and the resulting protein was purified. Enzyme assays and product characterization were used to show that the resulting recombinant protein is indeed dephosphoCoA kinase. The dephospho-CoA kinase gene is now designated coaE, based on the previous naming of the pantothenate kinase gene as coaA and of the phosphopantetheine adenylyltransferase gene as coaD (3, 7). MATERIALS AND METHODSMaterials. Dephospho-CoA, dilithium CoA, NADH, pyruvate kinase, lactic dehydrogenase, phosphoenolpyruvate, ATP, AMP, adenosine phosphosulfate (APS), DEAE Sepharose, and Q Sepharose were purchased from Sigma. A Sep...

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“…Therefore, as for B. ammoniagenes (15), the last two steps of CoA biosynthesis in E. coli are catalyzed by separate enzymes. Since PPAT is part of a bifunctional enzyme complex (with dPCoA kinase) in mammalian systems (10) and of a multi-enzyme complex (with among others dPCoA kinase) in bakers' yeast (14), there seems to be a clear difference in the organization of the enzymes of CoA biosynthesis in prokaryotic and eukaryotic organisms.…”

Section: Identification and Removal Of The Bound Compound(s) Frommentioning

confidence: 99%

“…Limited proteolysis of the latter revealed that the subunits are identical and that each subunit contains both PPAT and dPCoA kinase (11). Although similar bi-functional complexes have been partially purified from other mammalian sources (12,13), the PPAT of bakers' yeast (14) was identified as part of a much larger (375-400 kDa) CoA-synthesizing protein complex, which contained six different enzyme activities involved in the synthesis and metabolism of CoA. The complex could be separated into two components, the smallest of which (10 -15 kDa) contained both PPAT and dPCoA kinase activities.…”

mentioning

confidence: 98%

Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

Geerlof

Lewendon

Shaw³

1999

Journal of Biological Chemistry

111

112

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Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4-phosphopantetheine yielding 3-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.Coenzyme A (CoA) 1 is an essential cofactor in numerous biosynthetic, degradative, and energy-yielding metabolic pathways and is involved in the control of several key reactions in intermediary metabolism (1). CoA also donates the 4Ј-phosphopantetheinyl cofactor to the acyl carrier protein of the fatty acid synthase complex (2).The synthesis of CoA occurs in five steps which, utilize pantothenate (vitamin B 5 ), cysteine, and ATP (for review, see Ref.3). In all systems studied, the rate of CoA biosynthesis appears to be regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (4 -7). In vitro studies of pantothenate kinase from Escherichia coli showed that (a) CoA and, to a lesser extent, its acyl thioesters are competitive inhibitors with respect to ATP and (b) the K i values are within the physiological range of intracellular CoA concentrations (6). Studies of the intermediates in CoA biosynthesis have shown that both pantothenate and 4Ј-phosphopantetheine can accumulate in the cell (8). Hence, in addition to control of CoA synthesis on the level of pantothenate kinase, further modulation of flux through the pathway could occur at phosphopantetheine adenylyltransferase (PPAT), which catalyzes the penultimate step in the pathway (Fig. 1), the reversible adenylation of 4Ј-phosphopantetheine to form 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (PP i ). Regulation at this step may control the reutilization of 4Ј-phosphopantetheine arising either from the turnover of the 4Ј-phosphopantetheinyl cofactor of the acyl carrier protein (8) or the cleavage of CoA by a phosphodiesterase (9).Despite the above arguments for a role in the regulation of CoA biosynthesis, PPAT has not been the subject of a detailed study. Enzymes with PPAT activity have been purified from a number of different organisms. In mammals PPAT has been shown to be part of a complex that also includes dPCoA kinase, the effector of the fina...

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The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in baker's yeast

Cited by 21 publications

References 12 publications

The Yeast Mitochondrial Carrier Leu5p and Its Human Homologue Graves' Disease Protein Are Required for Accumulation of Coenzyme A in the Matrix

The Yeast Mitochondrial Carrier Leu5p and Its Human Homologue Graves' Disease Protein Are Required for Accumulation of Coenzyme A in the Matrix

Identification of yacE ( coaE ) as the Structural Gene for Dephosphocoenzyme A Kinase in Escherichia coli K-12

Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

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