Cloning and Characterization of a Eukaryotic Pantothenate Kinase Gene (panK) from Aspergillus nidulans

Abstract

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene fromEscherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designatedpanK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart. Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene fromEscherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designatedpanK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart. pantothenate kinase A. nidulans pantothenate kinase Saccharomyces cerevisiae pantothenate kinase Escherichia coli (bacterial) pantothenate kinase kilobases polymerase chain reaction isopropyl-thio-β-d-galactopyranoside. Pantothenate kinase (PanK)1(ATP:d-pantothenate 4′-phosphotransferase, EC 2.7.1.33) catalyzes the first committed step in the universal biosynthetic pathway leading to CoA. Phosphopantothenate is metabolized rapidly to CoA (for review, see Ref. 1Jackowski S. Neidhardt F.C. Curtiss R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar), which participates as an acyl group carrier in the tricarboxylic acid cycle, fatty acid metabolism, and numerous other reactions of intermediary metabolism (2Abiko Y. Greenburg D.M. Metabolic Pathways. Academic Press, New York1975: 1-25Google Scholar). The 4′-phosphopantetheine portion of CoA is an essential prosthetic group in a number of enzyme systems including the acyl carrier protein components of bacterial and eukaryotic fatty acid synthases (3Vagelos P.R. Curr. Top. Cell Regul. 1971; 4: 119-167Crossref Scopus (44) Google Scholar), citrate lyase (4Dimroth P. Eur. J. Biochem. 1976; 64: 269-281Crossref PubMed Scopus (25) Google Scholar), ferrichrome synthetase from Aspergillus quadricinctus (5Siegmund K.D. Plattner H.J. Diekmann H. Biochim. Biophys. Acta. 1991; 1076: 123-129Crossref PubMed Scopus (9) Google Scholar), and malonate decarboxylase of Malonomonas rubra (6Dimroth P. Hilbi H. Mol. Microbiol. 1998; 25: 3-10Crossref Scopus (39) Google Scholar). 4′-Phosphopantetheine is also required for δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase, the first enzyme for penicillin biosynthesis in fungi including Aspergillus nidulans (7MacCabe A.P. van Liempt H. Palissa H. Unkles S.E. Riach M.B.R. Pfeifer E. von Döhren H. Kinghorn J.R. J. Biol. Chem. 1991; 266: 12646-12654Abstract Full Text PDF PubMed Google Scholar). Escherichia coli is capable of de novo pantothenate biosynthesis, and a sodium-dependent permease actively transports pantothenate into the cell in both bacteria (8Vallari D.S. Rock C.O. J. Bacteriol. 1985; 162: 1156-1161Crossref PubMed Google Scholar, 9Vallari D.S. Rock C.O. J. Bacteriol. 1985; 164: 136-142Crossref PubMed Google Scholar, 10Jackowski S. Alix J.-H. J. Bacteriol. 1990; 172: 3842-3848Crossref PubMed Google Scholar) and mammals (11Lopaschuk G.D. Michalak M. Tsang H. J. Biol. Chem. 1987; 262: 3615-3619Abstract Full Text PDF PubMed Google Scholar,12Barbarat B. Podevin R.A. J. Biol. Chem. 1986; 261: 14455-14460Abstract Full Text PDF PubMed Google Scholar). However, metabolic labeling experiments in E. coli (13Jackowski S. Rock C.O. J. Bacteriol. 1981; 148: 926-932Crossref PubMed Google Scholar) and rat heart (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar, 15Robishaw J.D. Neely J.R. Am. J. Physiol. 1984; 246: H532-H541Crossref PubMed Google Scholar) show that the utilization, rather than the supply, of pantothenate controls the rate of CoA biosynthesis. In fact,E. coli produces 15-fold more pantothenate than is required for maintaining the intracellular CoA level (13Jackowski S. Rock C.O. J. Bacteriol. 1981; 148: 926-932Crossref PubMed Google Scholar). This excess pantothenate is excreted into the medium. E. coli mutants with temperature-sensitive bPanK activity are also temperature-sensitive for CoA biosynthesis and growth (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar). The bPanK gene of E. coli (coaA) was cloned by functional complementation and found to be identical to a previously sequenced temperature-sensitive allele called rts (17Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 1705-1706Crossref PubMed Google Scholar, 18Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 6411-6417Crossref PubMed Google Scholar, 19Flamm J.A. Friesen J.D. Otsuka J.A. Gene (Amst .). 1988; 74: 555-558Crossref PubMed Scopus (6) Google Scholar).E. coli bPanK is a homodimer of 36 kDa subunits which exhibits highly positive cooperative ATP binding and utilizes a sequential ordered mechanism with ATP as the leading substrate (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar). CoA and its thioesters inhibit bPanK activity by competitive binding to the ATP site (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar, 21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). Nonesterified CoA is the most potent inhibitor of bPanK in vitro and in vivo, whereas acetyl-CoA is about 20% as effective as CoA (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). CoA and CoA thioesters also inhibit mammalian (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar, 22Halvorsen O. Skrede S. Eur. J. Biochem. 1982; 124: 211-215Crossref PubMed Scopus (40) Google Scholar, 23Fisher M.N. Robishaw J.D. Neely J.R. J. Biol. Chem. 1985; 260: 15745-15751Abstract Full Text PDF PubMed Google Scholar) and plant (24Falk K.L. Guerra D.J. Arch. Biochem. Biophys. 1993; 301: 424-430Crossref PubMed Scopus (26) Google Scholar) PanK enzymes. Although both CoA and acetyl-CoA are reported to inhibit these enzymes, in general, acetyl-CoA is more effective than CoA. The goal of the present study was to extend the molecular and biochemical characterization of PanK to eukaryotic cells. Homologs ofE. coli bPanK protein and the coaA gene (accession no. M90071) are clearly detected in the genomes ofHemophilus influenzae (accession no. U32746),Mycobacterium tuberculosis (TIGR gmt7548), Vibrio cholerae (TIGR GVCCS17R), Streptococcus pyogenes(OUACGT Contig282), and Bacillus subtilis (accession no.D84432) using standard search and sequence alignment tools. In contrast, a similar search of the Saccharomyces cerevisiaegenome data base did not reveal the presence of a predicted protein with significant sequence similarity to the E. coli bPanK protein or the nucleotide sequence of the coaA gene. Also, sequences related to bPanK could not be identified in the mammalian expressed sequence-tagged data base. These results indicated that eukaryotic cells possess a PanK with a significantly dissimilar primary structure. Therefore, we employed a genetic selection strategy to clone a PanK gene from an A. nidulans cDNA library by functional complementation of the coaA15(Ts) mutant ofE. coli. This paper describes the isolation and characterization of a eukaryotic PanK with a distinctly different primary structure and dissimilar regulatory properties compared with its prokaryotic counterpart. Sources of supplies were: American Radiolabeled Chemicals, d-[1-14C]pantothenate (specific activity, 55 mCi/mmol); Appligene, pUC18; Bio-Rad, Bradford dye-binding protein assay solution; Boehringer Mannheim, Klenow fragment; Analtech Inc., 250-μm Silica Gel H plates; Bio 101, GeneClean II kit; NEN Life Science Products, d-[1-14C]pantothenate (specific activity, 54.5 mCi/mmol) and [α-32P]-dCTP (specific activity, 3000 Ci/mmol); Fisher Scientific, Scintisafe 30%; Amersham Pharmacia Biotech, Quick-Prep mRNA purification kit; Promega, restriction endonucleases and T4 DNA ligase; Qiagen, P100 columns; Schleicher & Schuell, nitrocellulose; Sigma, CoA, acetyl-CoA and kit; other or The bacterial in of E. coli S. Biol. PubMed Scopus Google Scholar) was from the was and did not of the temperature-sensitive allele by the coaA gene E. coli PanK and CoA biosynthesis (17Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 1705-1706Crossref PubMed Google Scholar, 18Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 6411-6417Crossref PubMed Google Scholar). also a temperature-sensitive growth for of the in the was to and CoA of a in the that a temperature-sensitive protein (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar). was H.J. D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and of H.J. D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) with and required amino was to for The was A. nidulans cDNA library was to the using the library from was isolated from nidulans nidulans with as the and in the of (7MacCabe A.P. van Liempt H. Palissa H. Unkles S.E. Riach M.B.R. Pfeifer E. von Döhren H. Kinghorn J.R. J. Biol. Chem. 1991; 266: 12646-12654Abstract Full Text PDF PubMed Google Scholar). of the in of E. coli was with of the cDNA library and for the E. coli bPanK was the and positive A. nidulans was with and and the was from the and sequences by in The was using a GeneClean II and as for of a with and Klenow from A. nidulans with the in citrate and with or and to for The was with the of and in of the positive was using a P100 and with a number of restriction enzymes. DNA by and with from The with a from of the The genomic DNA and into that been with of the genomic DNA with the A. nidulans cDNA identified the clone as the genomic DNA to the gene The genomic DNA and cDNA sequences on both by DNA using an and a with a of and of as using The cDNA sequence of panK was by DNA the Molecular of of the A. nidulans the for in or as D.J. Biochim. Biophys. Acta. PubMed Google Scholar) with or a of was from as previously A.P. Riach M.B.R. Unkles S.E. Kinghorn J.R. J. 1990; PubMed Scopus Google Scholar) and mRNA using a mRNA purification was as previously A.P. Riach M.B.R. Unkles S.E. Kinghorn J.R. J. 1990; PubMed Scopus Google Scholar) using a of The panK cDNA gene was from The a restriction site the and an site The a site of the polymerase chain reaction was using cDNA polymerase and the was into the The was into E. coli cells was isolated from the of cells and with and the was by The was and with This was to E. coli and for the by The E. coli coaA gene bPanK was by from site the first and and also a that an binding site to the of a site for The was using the cDNA polymerase and the was into The was into cells growth on selection DNA was isolated from a of cells and with and The was and isolated by The DNA was into with This was to and for and by for the presence of in the isolated and to in the presence of and for of bPanK protein by from that the protein of the molecular These into for purification and DNA to the amino acid sequence of the bPanK protein by the to and in the presence of and for to a protein of the size as a of from the the in to of and to a of was to a of and was for a 3 by in a and The cells by the of and The cells for on and the was isolated by in a for 3 The aPanK or the bPanK protein was in step on a by the The was with by with of of and of binding The was with the cell for and into a The was with of the binding by of the binding protein was with of binding The aPanK or bPanK and and and using an cell by a The in the presence of The of the protein was by on and as previously (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). The PanK in cell as a of protein with respect to both and protein by the of Bradford Biochem. 1976; PubMed Scopus Google Scholar) with as a activity 55 ATP and of protein from a in a of The was for and the reaction was by a a that was in three of acid in to was by the in 3 of cDNA library was as from A. The library was into the E. coli which a rts allele S. Biol. PubMed Scopus Google Scholar) and growth for growth of the that library that the and into coli or the which expressed a temperature-sensitive bPanK (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar). The coaA genes are (17Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 1705-1706Crossref PubMed Google Scholar). the temperature-sensitive growth in both bacterial mutants on than the from with the positive the E. coli coaA gene. growth of on is of a CoA with the level of amino acid (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar, S. Rock C.O. J. Bacteriol. 1986; PubMed Scopus Google Scholar). Therefore, of with was and the for the growth on the that complementation was not by of the These data clearly indicated that the A. nidulans cDNA expressed from the functional of an pantothenate The Aspergillus cDNA in was to a of genomic A. nidulans from the as DNA was and with from the of from the first identified as and from chromosome was with a of restriction enzymes, and the by and with the a of the a from an of the DNA and identified the as the genomic sequences for the cDNA in The gene for A. nidulans pantothenate kinase (aPanK) was and the genomic gene was to The DNA sequence of the aPanK cDNA and a of the panK gene was on both The panK gene a interrupted by three of found in fungi Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene in Press, Scholar). The of the by of the genomic and cDNA Although we not the protein sequence for the is the first of the was an located of the predicted The size of the expressed aPanK protein as the site as the is located amino The site of is from the and is indicated by the in However, the could not be by of the genomic and cDNA sequences is a of in the genomic sequence is located from the of mRNA from A. that the panK gene was as a mRNA The size of the panK was with the size predicted from the of the genomic and cDNA sequences The in the in cells with or as the in the presence or of and the level of panK was that that nidulans not The predicted protein sequence of aPanK of amino with a predicted molecular of 46.9 This sequence was to a similarity search the S. cerevisiae genome data base. This search identified a predicted that of amino acid with a predicted molecular of The was identical and similar to the aPanK sequence on similarity the is predicted to the pantothenate kinase The aPanK and was in the which was significantly in We detected a PanK in the S. cerevisiae The similarity aPanK and the previously bPanK coli was bPanK is a protein of amino with a molecular of bPanK was identical and and similar to These on the of significant in the bPanK sequence to the bacterial protein with the the bPanK and to the of the ATP binding site in is a in bPanK required for the binding of both the ATP substrate and the CoA to the enzyme (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar). The in bPanK to in aPanK and in as indicated in that these be in nucleotide binding in the aPanK and The functional complementation by with the DNA sequence indicated that panK gene a functional This was by from with for PanK a temperature-sensitive bPanK (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar), from with the a PanK of with in a in PanK activity in the of the cells not These results with the complementation and that the protein expressed by was a functional The biochemical properties of aPanK in more the of the aPanK cDNA into the the protein in E. coli and the protein by as The aPanK protein was in a step and was from a of cells in The was the of the step of the cells. The aPanK of a The subunit molecular size of the was with the molecular 46.9 predicted from the cDNA The activity of the aPanK from to the standard using an ATP of The in vitro kinase reaction was a and and of the activity not not required for the and the of was the other the of or not activity in the standard assay could ATP in the with to of the reaction could to of in a of in a of in a The of the aPanK for the The of in is of However, are of that by ordered systems the are not & New York1975: P. PubMed Scopus (40) Google Scholar). This the kinase has a for of the of the other rate the is The is both are Although the the of to the of the and the The pantothenate ATP was and the ATP pantothenate was PanK activity from different has been reported to be inhibited by CoA or CoA thioesters (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar, 21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar, 22Halvorsen O. Skrede S. Eur. J. Biochem. 1982; 124: 211-215Crossref PubMed Scopus (40) Google Scholar, 23Fisher M.N. Robishaw J.D. Neely J.R. J. Biol. Chem. 1985; 260: 15745-15751Abstract Full Text PDF PubMed Google Scholar). We found that acetyl-CoA was the most potent of the aPanK and we the of inhibition of enzyme activity and The of pantothenate activity indicated that acetyl-CoA inhibited the pantothenate and enzyme in a In these acetyl-CoA pantothenate kinase activity by ATP. In contrast, the of ATP with the enzyme was inhibited in a competitive by acetyl-CoA Acetyl-CoA activity by The for acetyl-CoA was as from the of the from these of inhibition by acetyl-CoA. aPanK was for activity with of pantothenate inhibitor or in the presence of or acetyl-CoA aPanK was also for activity with of ATP inhibitor or in the presence of or acetyl-CoA indicated that inhibition of aPanK by acetyl-CoA was with respect to pantothenate and competitive with respect to The mechanism of inhibition by a CoA was the in both the pantothenate from Aspergillus and coli (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar, 21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar) in that the inhibitor was competitive with respect to the ATP However, the pantothenate in to CoA or acetyl-CoA. We found previously that bPanK was inhibited potently by CoA (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). Acetyl-CoA and inhibited the bacterial enzyme to a lesser and was (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). Acetyl-CoA been reported to inhibit the mammalian pantothenate kinase activity, whereas CoA or other CoA thioesters little or (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar, 22Halvorsen O. Skrede S. Eur. J. Biochem. 1982; 124: 211-215Crossref PubMed Scopus (40) Google Scholar, 23Fisher M.N. Robishaw J.D. Neely J.R. J. Biol. Chem. 1985; 260: 15745-15751Abstract Full Text PDF PubMed Google Scholar). We the of the aPanK by CoA and its thioesters and compared it with of the bPanK from E. coli and ATP CoA to of aPanK by CoA was not whereas acetyl-CoA was effective the other bPanK was inhibited most potently by CoA and acetyl-CoA was activity by the of inhibitor was not a potent inhibitor of aPanK or bPanK ATP These data indicated that the ATP binding site of aPanK a distinctly different that selectively with the of acetyl-CoA as as the of ATP or CoA. The of similarity the primary of aPanK and the predicted nucleotide binding site was with these The of the protein sequence for a eukaryotic PanK the and molecular characterization of the PanK from The eukaryotic of PanK in has a primary structure that little resemblance to the previously enzyme expressed in prokaryotic cells. The bPanK and are that a of the eukaryotic PanK in the S. cerevisiae genome is not using standard search with bPanK as the the eukaryotic aPanK the reaction as and its to the E. coli mutants a temperature-sensitive bPanK the isolation of the aPanK the bPanK and the sequences by the of significant in the bPanK sequence in its alignment with aPanK the in nucleotide binding in bPanK (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar) to be in aPanK and However, of be essential the sequences in bPanK significantly from the sequences the in the and of is similar to a sequence in which is to of the ATP binding H.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). sequence also be of the ATP site in it is from The of the other of these The in the primary sequence of bPanK and aPanK the distinct in the regulatory properties of PanK coli and A. CoA and its thioesters inhibit bPanK activity by the binding of ATP to the enzyme (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar, 21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). In contrast, aPanK is regulated selectively by acetyl-CoA also in a competitive The by by different CoA molecular to the in CoA and coli is capable of pantothenate S. Neidhardt F.C. Curtiss R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar) and in produces and more pantothenate than it for CoA biosynthesis (13Jackowski S. Rock C.O. J. Bacteriol. 1981; 148: 926-932Crossref PubMed Google Scholar). Acetyl-CoA is an of metabolism and is in E. coli on as a (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). Thus, the of bPanK to acetyl-CoA to and the metabolic of rapidly cells. In contrast, and are to the of CoA and its thioesters in S. O. Biochem. Biophys. PubMed Scopus (25) Google Scholar, Biochem. J. 164: PubMed Scopus Google Scholar, A. Biochem. Biophys. 1981; PubMed Scopus Google Scholar, S. M. J. Biochem. 1986; PubMed Scopus Google Scholar, J.D. Neely J.R. Am. J. Physiol. 1985; PubMed Google Scholar). The PanK which is also in mammalian CoA biosynthesis (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar), is (14Robishaw J.D. Berkich D. Neely J.R. J. Biol. Chem. 1982; 257: 10967-10972Abstract Full Text PDF PubMed Google Scholar, 22Halvorsen O. Skrede S. Eur. J. Biochem. 1982; 124: 211-215Crossref PubMed Scopus (40) Google Scholar, 23Fisher M.N. Robishaw J.D. Neely J.R. J. Biol. Chem. 1985; 260: 15745-15751Abstract Full Text PDF PubMed Google Scholar). Thus, PanK is not located in the to a significant of the CoA of to acetyl-CoA to a mechanism for of CoA to the of CoA by activity of key regulatory The of aPanK for acetyl-CoA that the ATP binding site is distinctly different from that found in bPanK or other enzymes. The amino the in the ATP binding site in aPanK not a or a nucleotide binding site M. J. 1982; PubMed Scopus Google Scholar) and a unique ATP binding of the structure of bPanK with that of aPanK reveal the that the for the as as of the which identical We A. J. of for A. nidulans We also of for