Coenzyme a-synthesizing protein complex of Saccharomyces cerevisiae

Bucovaz, E.T.; Tarnowski, S.Joseph; Morrison, William C.; MacLeod, Robert M.; Morrison, John C.; Sobhy, C.M.; Rhoades, James L.; Fryer, James E.; Wakim, Jubran M.; Whybrew, W.D.

doi:10.1007/bf00215301

Cited by 15 publications

(12 citation statements)

References 15 publications

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“…Previous biochemical data prompted the assertion that an alternate CoA biosynthetic pathway exists in S. cerevisiae (30). However, the identification of yeast orthologs for all of the human CoA biosynthetic enzymes described here, in combination with the essentiality of the corresponding S. cerevisiae genes, suggests that the CoA biosynthetic route in yeast is similar to that in E. coli and humans and that the former interpretation needs to be revised.…”

Section: Discussionmentioning

confidence: 77%

Complete Reconstitution of the Human Coenzyme A Biosynthetic Pathway via Comparative Genomics

Daugherty¹,

Polanuyer²,

Farrell³

et al. 2002

Journal of Biological Chemistry

205

181

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The biosynthesis of CoA from pantothenic acid (vitamin B 5 ) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC 6.3.2.5), phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36), phosphopantetheine adenylyltransferase (EC 2.7.7.3), and dephospho-CoA kinase (EC 2.7.1.24). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.

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Section: Discussionmentioning

confidence: 77%

Complete Reconstitution of the Human Coenzyme A Biosynthetic Pathway via Comparative Genomics

Daugherty¹,

Polanuyer²,

Farrell³

et al. 2002

Journal of Biological Chemistry

205

181

View full text Add to dashboard Cite

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“…These differences suggest the existence of distinct modes of regulation of CoA biosynthesis. Indeed, the CoA-synthesizing complex in S. cerevisiae was found to have a molecular mass around 400 kDa, while in higher eukaryotes, the existence of such a complex has not been reported (23). The compartmentalization of the CoA biosynthetic pathway is poorly understood in mammals.…”

Section: Discussionmentioning

confidence: 99%

“…The reaction mixture was incubated at 25°C for 30 min. Reaction products were separated by descending paper chromatography using Whatman 3MM paper and developing system containing isobutyric acid:0.5 N ammonium hydroxide (100:60) and 1 mM EDTA (23). A phosphoimager system (Bio-Rad) was used to identify the position of radiolabelled products.…”

Section: Methodsmentioning

confidence: 99%

Molecular Cloning of CoA Synthase

Zhyvoloup¹,

Nemazanyy²,

Бабич³

et al. 2002

Journal of Biological Chemistry

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Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of ϳ60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.

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“…Therefore, S. cerevisiae's PPCDC is not a monogenic enzyme, but instead consist of at least two different gene products that are not functionally exchangeable. Previous work has suggested the existence of a 375-400 kDa CoA biosynthesizing complex in S. cerevisiae that would involve an alternative pathway for the biosynthesis of CoA (with a decarboxylase enzyme acting on 5′-ADP-4′-pantothenoylcysteine (10) to form dephospho-CoA (11)) 42,43 . In this regard, we wish to stress that: 1) In spite of the many reported genome-wide experiments to identify protein-protein interactions in S. cerevisiae, no interaction has been described between any of the putative CoA biosynthetic proteins (except for interactions of Ykl088w with Hal3 and Vhs3), suggesting that the heteromeric PPCDC described here is not related to the complex mentioned above.…”

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confidence: 99%

Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis

et al. 2009

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Unlike most other organisms, the essential five-step Coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the gene(s) encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remains unidentified.Sequence homology analyses suggest three candidates, namely Ykl088w, Hal3 and Vhs3, as putative PPCDC enzymes in Saccharomyces cerevisiae. Interestingly, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3/Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer which consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues.Our results unveil Hal3/Vhs3 as moonlighting proteins, involved in both CoA biosynthesis and protein phosphatase regulation. 3Coenzyme A (CoA, 1) is a ubiquitous and essential cofactor that is utilized by a wide variety of enzymes in reactions where it mainly acts as a carrier and activator of acyl groups 1, 2 . The CoA biosynthetic pathway has been elucidated in various diverse species, including eubacteria (Escherichia coli), plants (Arabidopsis thaliana), and mammals (Homo sapiens) 1,3,4 . These studies have shown that the pathway is universal and consists of the same five enzymatic transformations in all cases, although some diversity exist among the specific proteins that catalyze certain steps 5 . Nonetheless, bioinformatic approaches allow identifying (with a few exceptions 6 ) candidate genes encoding the CoA biosynthetic proteins in nearly all organisms, including Saccharomyces cerevisiae.Interestingly, sequence homology searches suggest three proteins, namely Hal3, Vhs3 and Ykl088w, as candidates that may exhibit phosphopantothenoylcysteine decarboxylase (PPCDC) activity in S. cerevisiae. PPCDC is a flavoprotein that catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine (PPC; 2) to yield 4'-phosphopantetheine (PP; 3), the third step in CoA biosynthesis. While previous studies on PPCDCs have shown some diversity among these enzymes (e.g. the bacterial variants are usually bifunctional proteins that also have phosphopantothenoylcysteine synthetase (PPCS) activity), they all share a common mechanism and active site architecture [7][8][9][10][11] . Furthermore, all PPCDCs characterized so far are monogenic. While the three PPCDC candidates in S. cerevisiae are indeed very similar (Vhs3 and Ykl088w have 49% and 28% sequence identity to the Hal3 protein respectively, see Figure 1a), the identification of Hal3 and Vhs3 as potential PPCDCs is in fact surprising as both proteins have previously been shown to have functions completely unrelated to CoA biosynthesis.Specifically, Hal3 (also known as Sis2) is a conserved protein originally identified as a halotoler...

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Coenzyme a-synthesizing protein complex of Saccharomyces cerevisiae

Cited by 15 publications

References 15 publications

Complete Reconstitution of the Human Coenzyme A Biosynthetic Pathway via Comparative Genomics

Complete Reconstitution of the Human Coenzyme A Biosynthetic Pathway via Comparative Genomics

Molecular Cloning of CoA Synthase

Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis

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