Cloning and Characterization of a Eukaryotic Pantothenate Kinase Gene (panK) from Aspergillus nidulans
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2014
ISSN1083-351X
AutoresRobert B. Calder, Robin S. B. Williams, Gayathri Ramaswamy, Charles O. Rock, Eddie Campbell, Shiela E. Unkles, James R. Kinghorn, Suzanne Jackowski,
Tópico(s)Cancer-related gene regulation
ResumoPantothenate 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 malonyl-CoA; Stratagene, ZAP-cDNA synthesis kit; Whatman, DE81 filter circles. All other materials were reagent grade or better. The bacterial strains used in this work were derivatives of E. coli K-12. Strain ts9 (leuB6 hisG1 argG6 metB1 rplL9 rts-1 ilu-1 lacY1 gal-6 xyl-7 mtl-2 malA1 tonA2 tsx-1 λRλ− supE44) (25Flaks J.G. Leboy S. Birge E.A. Kurland C.G. Cold Spring Harbor Symp. Quant. Biol. 1966; 31: 623-631Crossref PubMed Scopus (29) Google Scholar) was obtained from the Coli Genetic Stock Center, Yale University. Strain ts9 was conditionally defective and did not grow at 42 °C. Complementation of the temperature-sensitive rts-1 allele by the coaA gene (pWS7-13-2) encoding E. coli PanK permitted phosphopantothenate 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). Strain ts9 also exhibited a temperature-sensitive growth requirement for isoleucine because of the defect in the ilv-1 allele. Strain DV73 (coaA15 srl::Tn10 recA metB1 relA1 spoT1 gyrA216 λ− λRF−) was unable to synthesize phosphopantothenate and CoA at 42 °C because of a specific defect in the coaA15allele that encoded a temperature-sensitive (bPanK) protein (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar). Rich medium was Luria broth (26Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar), and minimal medium consisted of medium E salts (26Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar) supplemented with glucose (0.4%), thiamine (0.001%), and required amino acids (0.01%). Agar (1.5%) was added to media formulations for plating. The ampicillin concentration was 50 μg/ml when indicated. An A. nidulans cDNA library was prepared according to the manufacturer's instructions using the ZAP-cDNA library kit from Stratagene. Messenger RNA (5.1 μg) was isolated from wild-typeA. nidulans strain G1071 (completely prototrophic) grown onA. nidulans minimal medium with 10 mm sodium nitrate as the sole nitrogen source and in the absence of vitamins (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). Excision of the λ-ZAP yielded 2.5 × 108 phagemids in 30 μg of DNA. E. coli strain ts9 was electroporated (2,500 volts, capacitance 25 microfarads, pulse time 4.88 ms) with 0.5 μg of the excised cDNA library and incubated at 23 °C for 2 h. Transformants were plated onto Luria broth agar plus ampicillin at 37 °C. Plasmid pWS7-13-2 encoding the E. coli bPanK was the transformation and phenotypic positive control. Recombinant A. nidulans phagemid λSTA1999 was digested with KpnI and SpeI, and the 450-base pair fragment was separated from the 1.5-kb and 2.9-kb vector sequences by gel electrophoresis in 1% agarose. The fragment was purified using a GeneClean II kit and then used as template for preparation of a radiolabeled probe together with [32P]dCTP, dATP, dTTP, dGTP, and Klenow fragment. Southern blots prepared from A. nidulans chromosomes I–VIII (Fungal Genetic Stock Center) were hybridized with the probe. Blots were washed at 25 °C in 2 × saline sodium citrate and exposed overnight at −70 °C. Hybridizing cosmids were purified, digested with EcoRV, EcoRV plus XhoI, or BamHI plus BglII, and transferred to Zeta-Probe membrane for rescreening. The membrane was then hybridized with the KpnI/SpeI fragment of phagemid λSTA1999 and washed at 65 °C in 1 × saline sodium citrate. One of the four positive cosmids, W19B06 (34C5), was purified using a Qiagen P100 column and digested with a number of restriction enzymes. Genomic DNA fragments were separated by agarose gel electrophoresis, blotted onto Zeta-Probe membrane, and probed with theKpnI/SpeI fragment derived from phagemid λSTA1999. The probe hybridized with a 5.0/5.1-kb doublet band from anXbaI digest of the cosmid. The two genomic DNA fragments were separated, purified, and subcloned into pUC18 that had been digested with XbaI. Hybridization of the two genomic DNA fragments with the A. nidulans phagemid λSTA1999 cDNA fragment identified the 5.1-kb clone (plasmid pSTA2000) as containing the genomic DNA corresponding to the gene designated panK. The genomic DNA (pSTA2000) and cDNA (λSTA1999) sequences were determined on both strands by automated DNA sequencing using an Applied Biosystems 373A automated fluorescent sequencing apparatus and a PRISM Ready Reaction dideoxy terminator cycle sequencing kit (Applied Biosystems) with primers at a concentration of 10 μm and 0.5 μg of pSTA2000 as template. Sequences were assembled using Sequencher (Gene Codes Corp.). The cDNA sequence of panK was verified independently by automated DNA sequencing at the Molecular Resource Center of St. Jude Children's Research Hospital. Cultures of the wild-type A. nidulans strain GO51 (carrying the biotin auxotrophic markerbia1) were grown at 30 °C for 16 h in liquid minimal medium containing 5 mm ammonium tartrate or 10 mm sodium nitrate as sole nitrogen source (27Cove D.J. Biochim. Biophys. Acta. 1966; 113: 51-56Crossref PubMed Google Scholar) with or without a final concentration of 1 μg/ml pantothenate. Total RNA was extracted from mycelium as described previously (28MacCabe A.P. Riach M.B.R. Unkles S.E. Kinghorn J.R. EMBO J. 1990; 9: 279-287Crossref PubMed Scopus (102) Google Scholar) and mRNA prepared using a Quick Prep mRNA purification kit. Northern blot analysis was carried out as described previously (28MacCabe A.P. Riach M.B.R. Unkles S.E. Kinghorn J.R. EMBO J. 1990; 9: 279-287Crossref PubMed Scopus (102) Google Scholar) using a 2-kbBamHI fragment of pSTA2000. The panK cDNA gene fromAspergillus was amplified from phagemid λSTA1999, encoding aPanK. The forward primer created a novel restriction site forNdeI at the amino-terminal methionine and removed an internal BamHI site (5′-GTCATATGTCCGCCACTGATCCTACTC-3′). The reverse primer introduced a BamHI site downstream of the stop codon (5′-AGGATCCGGTTGCCGCCTAAGCTCAT-3′). A polymerase chain reaction (PCR) was performed using Advantage cDNA polymerase mix (CLONTECH), and the product was ligated into the TA cloning vector pCR2.1 (Invitrogen). The ligation mixture was transformed into E. coli One Shot cells (Invitrogen). After overnight growth, plasmid was isolated from the ampicillin-resistant population of cells and digested with NdeI andBamHI, and the appropriate fragment was gel purified by QIAquick (Qiagen). The purified fragment was ligated intoNdeI and BamHI digested pET-15b (Novagen) treated with alkaline calf intestinal phosphatase. This ligation mixture was used to transform E. coli strain BL21(DE3) (Novagen), and ampicillin-resistant transformants were screened for the correct insertion by PCR. The E. coli coaA gene encoding bPanK was amplified by PCR from pWS7-13-2. A forward primer (5′-CATATGAGTATAAAAGATCAAACG-3′) introduced aNdeI site at the first translational start and and also introduced a mutation (E5D) that removed an internal ribosomal binding site to reduce the occurrence of shorter transcripts. A reverse primer (5′-GGATCCGAGTATTCGCTCCCCTGCAA-3′) added a BamHI site for subcloning. The PCR was performed using the Advantage cDNA polymerase mix (CLONTECH), and the product was ligated into pCR2.1 (Invitrogen). The ligation mixture was transformed into One Shot cells (Invitrogen). After overnight growth on ampicillin selection medium, plasmid DNA was isolated from a mixture of cells and digested with NdeI and BamHI. The appropriate fragment was gel purified and isolated by QIAquick (Qiagen). The purified DNA fragment was ligated into NdeI- andBamHI-digested pET-15b (Novagen) treated with calf intestinal alkaline phosphatase. This ligation mixture was used to transform strain BL21(DE3) (Novagen), and transformants were screened for ampicillin resistance and by PCR for the presence of thecoaA insert in the plasmid vector. Single colonies were isolated and cultured to mid-log phase, frozen at −70 °C in the presence of 7% dimethyl sulfoxide, and screened for overexpression of bPanK protein by SDS-polyacrylamide gel electrophoresis after IPTG induction. Plasmids were recovered from several clonal cultures that overexpressed the His-tagged protein of the appropriate molecular size. These plasmids were each transformed into strain DH5α for subsequent plasmid purification and DNA sequencing to verify the amino acid sequence of the bPanK protein encoded by the plasmid. Colonies were grown to mid-log phase and frozen at −70 °C in the presence of 7% dimethyl sulfoxide and tested for their ability to overexpress a protein of the appropriate size after IPTG induction. Overnight cultures were prepared as a series of 1:100 dilutions made directly from the thawed freeze down; the following morning, cultures in early-mid log phase were added to 500 ml of LB and grown to a density of approximately 5 × 108cells/ml. IPTG was then added to a final concentration of 1 mm, and incubation was continued for a further 3 h at 37 °C. Cells were collected by centrifugation in a JA-10 rotor (8,000 rpm, 4 °C, 10 min) and stored at −20 °C overnight. The cells were lysed by the addition of 1 mg/ml DNase and lysozyme plus 0.1% Triton X-100. The cells were then frozen at −70 °C for 2 h, thawed on ice, and the soluble extract was isolated by centrifugation in a Ti 45 rotor at 40,000 rpm for 3 h. The aPanK or the bPanK His-tagged fusion protein was purified in one step on a Ni-NTA agarose resin (Qiagen) by the same procedure. The affinity matrix (15 ml) was charged with NiSO4 by sequentially washing with 60 ml of water, 60 ml of 2 mmNiSO4, and 90 ml of binding buffer (20 mmTris-HCl, pH 7.8, plus 0.5 m NaCl). The activated support was mixed with the cell extract for 45 min at 4 °C and then packed into a column. The column was washed successively with 150 ml of the binding buffer followed by 150 ml of the binding buffer containing 0.04m imidazole. Bound protein was eluted with 150 ml of binding buffer containing 0.2 m imidazole. The fractions containing His-tagged aPanK or bPanK were combined and dialyzed against 20 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, and 1 mm EDTA and concentrated using an Amicon stirred cell followed by a Centricon-30 concentrator. The purified proteins were stored at −20 °C in the presence of 50% glycerol. The purity of the protein preparations was assessed by SDS-gel electrophoresis on 12% polyacrylamide gels. Enzyme preparation and assays were performed as described previously (21Vallari D.S. Jackowski S. Rock C.O. J. Biol. Chem. 1987; 262: 2468-2471Abstract Full Text PDF PubMed Google Scholar). The PanK specific activities in cell lysates were calculated as a function of protein concentration. Assays were linear with respect to both time and protein input. Protein concentrations were measured by the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214321) Google Scholar) with γ-globulin as a standard. Standard assays containedd-[1-14C]pantothenate (45.5 μm; specific activity 55 mCi/mmol), ATP (2.5 mm, pH 7.0), MgCl2 (2.5 mm), Tris-HCl (0.1 m, pH 7.5), and 32 μg of protein from a 35–60% ammonium sulfate fraction in a total volume of 40 μl. The mixture was incubated for 10 min at 37 °C, and the reaction was stopped by depositing a 30-μl aliquot onto a Whatmann DE81 ion exchange filter disc that was washed in three changes of 1% acetic acid in 95% ethanol (25 ml/disc) to remove unreacted pantothenate. 4′-Phosphopantothenate was quantitated by counting the dried disk in 3 ml of scintillation solution. A cDNA expression library was prepared as described under "Experimental Procedures" from wild-type A. nidulans, prototrophic strain G1071. The library was transformed into the E. coli strain ts9, which carried a conditionally defective rts allele (25Flaks J.G. Leboy S. Birge E.A. Kurland C.G. Cold Spring Harbor Symp. Quant. Biol. 1966; 31: 623-631Crossref PubMed Scopus (29) Google Scholar) and exhibited poor growth at 37 °C. Selection for growth of the transformants at 37 °C yielded colonies that harbored library phagemids that functionally complemented the defect. Three phagemids were purified and retransformed into eitherE. coli strains ts9 or DV73. Strain DV73 harbored the defective coaA15 allele, which expressed a temperature-sensitive bPanK (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar). The coaA andrts genes are allelic (17Song W.-J. Jackowski S. J. Bacteriol. 1992; 174: 1705-1706Crossref PubMed Google Scholar). Phagemid designated λSTA1999 complemented the temperature-sensitive growth defect in both bacterial mutants on rich medium, albeit yielding slightly smaller colony diameters than the control colonies arising from transformation with the positive control plasmid, pWS7-13-2, expressing the E. coli coaA gene. Limited growth of strain DV73 (coaA15(Ts)) on rich medium at 42 °C is often observed because of a large preexisting CoA pool coupled with the high level of amino acid supplementation (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar, 30Jackowski S. Rock C.O. J. Bacteriol. 1986; 166: 866-871Crossref PubMed Scopus (64) Google Scholar). Therefore, transformation of strain DV73 with phagemid λSTA1999 was repeated, and ampicillin-resistant colonies were selected at the permissive temperature, 30 °C. Subsequently, 48 colonies were scored for the temperature-dependent growth phenotype on glucose-minimal medium at 42 °C. All 48 colonies grew at the nonpermissive temperature, verifying that complementation was not caused by reversion of the host strain phenotype. These data clearly indicated that the A. nidulans cDNA expressed from phagemid λSTA1999 encoded the functional equivalent of an active pantothenate kinase. The Aspergillus cDNA insert in phagemid λSTA1999 was used to screen a bank of genomic clones representing A. nidulans chromosomes I–VIII obtained from the Fungal Genetic Stock Center as described under "Experimental Procedures." Genomic DNA was blotted onto membranes and hybridized with a32P-labeled probe derived from the 450-base pairKpnI/SpeI fragment of phagemid λSTA1999. Positive cosmids from the first screen were identified as W19B06, W21A12, W21H08, W23E02, W23D11, W24H12, and W24H03 from chromosome III. Cosmid W19B06 (34C5) was digested with a panel of restriction enzymes, and the fragments were separated by agarose gel electrophoresis and blotted onto membranes. Hybridization with the 450-base pair λSTA1999 probe signaled a region of the gel containing a 5.0/5.1-kb doublet from an XbaI digest. Purification of the two DNA bands and reprobing identified the 5.1-kb fragment as containing the genomic sequences for the cDNA insertion in phagemid λSTA1999. The gene for A. nidulans pantothenate kinase (aPanK) was designatedpanK, and the genomic fragment containing this gene was subcloned to yield plasmid pSTA2000. The DNA sequence of the aPanK cDNA and a 2115-base pair stretch of plasmid pSTA2000 encompassing the panK gene was determined on both strands (Fig. 1). The panK gene contained a single open reading frame interrupted by three short introns typical of those found in fungi (31Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford1987: 93-139Google Scholar). The positions of the introns were confirmed by comparison of the genomic and cDNA sequences. Although we have not directly determined the amino-terminal protein sequence for aPanK, the putative translational start is the first methionine codon of the open reading frame. There was an in-frame stop codon located 7 codons upstream of the predicted methionine start codon. The size of the expressed aPanK protein (see below) confirms this Met as the start site as the next methionine residue is located 154 amino acids downstream. The site of polyadenylation is approximately 508 nucleotides downstream from the translational stop codon and is indicated by the arrow in Fig. 1. However, the precise polyadenylation acceptor could not be determined simply by comparison of the genomic and cDNA sequences because there is a stretch of 6 A nucleotides in the genomic sequence at this point. An AATAAA polyadenylation signal is located 21 nucleotides upstream from the proposed polyadenylation site. Northern blot analysis of mRNA prepared from A. nidulansindicated that the panK gene was transcribed as a single mRNA (Fig. 2 A). The apparent size of the panK transcript, 1.85 kb, was consistent with the size predicted from the analysis of the genomic and cDNA sequences (Fig. 1). The transcript occurred in approximately the same abundance in cells grown with either ammonium or nitrate as the nitrogen source, in the presence or absence of pantothenate, and the level of panK transcript was much lower that that ofA. nidulans actin (data not shown). The predicted protein sequence of aPanK consisted of 420 amino acids with a predicted molecular mass of 46.9 kDa. This sequence was used to perform a similarity search against the S. cerevisiae genome data base. This search identified a predicted open reading frame (Ydr531w) that consisted of 367 amino acid residues with a predicted molecular mass of 40.9 kDa. The Ydr531w open reading frame was 44.8% identical and 60.2% similar to the aPanK sequence (Fig. 3). Based on this strong similarity the Ydr532w open reading frame is predicted to encode the yeast pantothenate kinase (yPanK). The major difference between aPanK and yPanK was in the amino-terminal domain, which was significantly longer in aPanK. We detected only a single PanK isoform in the S. cerevisiae genome. The similarity between aPanK and the previously described bPanK fromE. coli was far less striking (Fig. 3). bPanK is a protein composed of 316 amino acids with a molecular mass of 36.4 kDa. bPanK was 16.2% identical and and 32.9% similar to aPanK. These calculations were based on the introduction of several significant gaps in the bPanK sequence to align the smaller bacterial protein with the larger aPanK. Nonetheless, the comparison between bPanK and aPanK/yPanK points to the location of the ATP binding site in aPanK/yPanK. Lysine 101 is a critical residue in bPanK required for the binding of both the ATP substrate and the CoA regulators to the enzyme (20Song W.-J. Jackowski S. J. Biol. Chem. 1994; 269: 27051-27058Abstract Full Text PDF PubMed Google Scholar). The Lys-101 residue in bPanK corresponds to Lys-141 in aPanK and Lys-85 in yPanK as indicated in Fig. 3, suggesting that these lysine residues may be involved in nucleotide binding in the aPanK and yPanK proteins. The functional complementation by phagemid λSTA1999 coupled with the cDNA/genomic DNA sequence analysis strongly indicated that theApergillus panK gene encoded a functional PanK. This point was tested by assaying extracts from strain DV73 (coaA15(Ts)) transformed with phagemid λSTA1999 for PanK activity. Because strain DV73 had a temperature-sensitive bPanK (16Vallari D.S. Rock C.O. J. Bacteriol. 1987; 169: 5795-5800Crossref PubMed Google Scholar), extracts from strain DV73 transformed with the empty control plasmid possessed a low background PanK specific activity. Transformation of strain DV73 with phagemid λSTA1999 resulted in a dramatic increase in PanK activity in the soluble fractions of the cells (data not shown). These results were consistent wi
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