JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M306321200 on July 14, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38229-38237, October 3, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/40/38229    most recent
M306321200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kupke, T.
Right arrow Articles by Culiáñez-Macià, F. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kupke, T.
Right arrow Articles by Culiáñez-Macià, F. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

4'-Phosphopantetheine and Coenzyme A Biosynthesis in Plants*

Thomas Kupke {ddagger} §, Pilar Hernández-Acosta ¶ and Francisco A. Culiáñez-Macià ¶ ||

From the {ddagger}Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany and the Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, 46022 Valencia, Spain

Received for publication, June 16, 2003 , and in revised form, July 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Coenzyme A is required for many synthetic and degradative reactions in intermediary metabolism and is the principal acyl carrier in prokaryotic and eukaryotic cells. Coenzyme A is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes in bacteria and human have all been identified and characterized. Coenzyme A biosynthesis in plants is not fully understood, and to date only the AtHAL3a (AtCoaC) gene of Arabidopsis thaliana has been cloned and identified as 4'-phosphopantothenoylcysteine (PPC) decarboxylase (Kupke, T., Hernández-Acosta, P., Steinbacher, S., and Culiáñez-Macià, F. A. (2001) J. Biol. Chem. 276, 19190–19196). Here, we demonstrate the cloning of the four missing genes, purification of the enzymes, and identification of their functions. In contrast to bacterial PPC synthetases, the plant synthetase is not CTP-but ATP-dependent. The complete biosynthetic pathway from pantothenate to coenzyme A was reconstituted in vitro by adding the enzymes pantothenate kinase (AtCoaA), 4'-phosphopantothenoylcysteine synthetase (AtCoaB), 4'-phosphopantothenoylcysteine decarboxylase (AtCoaC), 4'-phosphopantetheine adenylyltransferase (AtCoaD), and dephospho-coenzyme A kinase (AtCoaE) to a mixture containing pantothenate, cysteine, ATP, dithiothreitol, and Mg2+.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Coenzyme A and 4'-phosphopantetheine are essential cofactors for many enzymatic reactions, and acyl-CoA derivatives are key intermediates in energy metabolism (1). In coenzyme A, 4'-phosphopantetheine is covalently linked to an adenylyl group, whereas it is covalently linked to a serine hydroxyl group in acyl carrier proteins. 4'-phosphopantetheine is cofactor of enzymes that play a role in the biosynthesis of fatty acids, polypeptide antibiotics, and polyketides (2). It was Feodor Lynen who elucidated that the thiol group of the cysteamine moiety of coenzyme A is the functional group by activating substrates as thioesters (3).

Coenzyme A is synthesized in five steps from pantothenate (vitamin B5) (4–6) and, despite its biological significance, only recently have all the biosynthetic enzymes been cloned and characterized in prokaryotes (7–11), and the human genes encoding the last four enzymatic steps in coenzyme A biosynthesis were identified last year (12). In the first step, pantothenate is phosphorylated to 4'-phosphopantothenate by pantothenate kinase, which is encoded by the coaA gene. Then, (R)-4'-phospho-N-pantothenoylcysteine (PPC)1 is synthesized by the addition of cysteine to 4'-phosphopantothenate and, in the next step, PPC is decarboxylated to 4'-phosphopantetheine. 4'-phosphopantetheine is converted to coenzyme A by the enzymes 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase (Fig. 1).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1.
The plant 4'-phosphopantetheine and coenzyme A biosynthetic pathway. Coenzyme A is synthesized from pantothenate in five steps. The A. thaliana enzymes AtCoaA, AtCoaB, AtCoaC, AtCoaD, and AtCoaE catalyze the phosphorylation of pantothenate, the ligation of cysteine and 4'-phosphopantothenate to 4'-phosphopantothenoylcysteine, the decarboxylation of PPC to 4'-phosphopantetheine, the formation of dephospho-coenyzme A, and the phosphorylation of the 3'-hydroxyl group of dephospho-coenzyme A. The previously characterized AtHAL3 is the AtCoaC enzyme. Names follow the prokaryotic nomenclature.

 

In plants, where coenzyme A metabolism is well understood (13), little is known about its biosynthesis (14). Recently, the Arabidopsis thaliana flavoprotein AtHAL3a, which shows sequence homology to the bacterial flavoprotein Dfp (8, 15), has been identified as the enzyme that catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, the third step in coenzyme A biosynthesis (16–18).

Bacterial and human sequences of coenzyme A biosynthetic genes are very different in some cases and, therefore, are interesting novel drug targets (12, 19). Recently, N-substituted pantothenamides have been identified as inhibitors of the staphylococcal pantothenate kinase (20), and dipeptides were used as inhibitors of the bacterial phosphopantetheine adenylyltransferase activity (21).

The complete sequence of the A. thaliana genome (22) gives the molecular biologists a magnificent tool for elucidating the function of plant genes. To gain further insight into the coenzyme A biosynthesis in plants, we identified the four missing A. thaliana coenzyme A biosynthetic genes via comparative genomics using the BLAST program (23) and the known sequences/conserved sequence motifs of prokaryotic and eukaryotic enzymes. According to the prokaryotic nomenclature, the biosynthetic genes of A. thaliana encoding the pantothenate kinase, the 4'-phosphopantothenoylcysteine synthetase, the 4'-phosphopantetheine adenylyltransferase, and the dephosphocoenzyme A kinase were named AtCoaA, AtCoaB, AtCoaD, and AtCoaE, respectively. The AtCoa genes were reverse transcriptase PCR amplified, and the cDNAs were cloned into bacterial expression vectors and over-expressed. The expressed recombinant proteins were purified, their activity was determined, and the entire plant coenzyme A biosynthesis pathway, starting with pantothenate, was reconstituted in vitro.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cloning of AtCoa Genes
Materials—The materials used for cloning were obtained from New England Biolabs (pMAL-c2X vector and Escherichia coli TB1 host for expression), Roche Applied Science (First Strand cDNA synthesis kit for reverse transcriptase PCR (avian myeloblastis virus)), Sigma (GenElute mammalian total RNA kit and REDTaq DNA polymerase), Sigma-Genosys (oligonucleotides), and Stratagene (pBluescript SK+ vector). The pSBETa helper vector was constructed at the Max-Planck Institut, Köln, Germany (24).

Plant Material—A. thaliana ecotype Columbia was grown in the greenhouse at 25 °C for 8 h in the dark and 16 h in light.

Genome Analysis—Comparative genomics was performed using programs such as BLAST (23) and data bank resources from the NCBI. Protein domain families were generated with the ProDom program from the Swiss-Prot and TrEMBL sequence databases (25). The Wisconsin Package software, Version 10.0-UNIX, from Genetics Computer Group (GCG; Madison, WI) was used for Pileup and Prettybox sequence alignments with gcg10.

cDNA Cloning—Using peptide sequence motifs shared between prokaryotic and eukaryotic CoA biosynthetic enzymes (i.e. the Walker kinase motif of E. coli CoaE; Refs. 26 and 27), virtual clones were isolated by BLAST (23) search screening from the predicted conceptual translated proteins of the A. thaliana genomic library. The corresponding homologous genes were cloned by reverse transcriptase PCR using leaf total RNA as template for the first strand cDNA synthesis together with an oligo(dT) primer and avian myeloblastis virus reverse transcriptase. The first strand cDNA template was PCR amplified using REDTaq DNA polymerase and the 5'-forward and 3'-reverse gene-specific adapted primers 5'-CGAGGAATTCATGGATCCGACTCAAATCTCTC-3'/5'-CCGGCTCGAGCTAAACTAAATTGATGCTACATTC-3'; 5'-CCGGGGATCCATGAGTTCGATCTCTGGATTGGTGGAAG-3'/5'-CCGGCTCGAGTCAAGTGAGAGATTCTTTGATGTATG-3'; 5'-CCGGGGATCCATGGCAGCTCCGGAAGATTCAAAGATG-3'/5-CGAGAAGC TTCCACCTCATGATGCTTTTTCTTCTGCTGGTTG-3'; and 5'-CCGGGGATCCATGAGAATAGTCGGGTTAACGGG-3'/5'-CGAGAAGCTTCCACCTTAAGAGCCAATTTTGAGCTGTTTGC-3' for AtCoaA, AtCoaB, AtCoaD, and AtCoaE, respectively. The PCR products were cloned into the pBluescript SK+ vector, and then the full-length cDNAs, containing the entire gene coding regions, were subcloned into the pMAL-c2X expression vector (EcoRI/SalI in the case of AtCoaA, BamHI/SalI in the case of AtCoaB, and BamHI/HindIII for AtCoaD and AtCoaE) and transformed into the expression strain E. coli TB1 for recombinant protein production. Depending on which cloning site of the pMAL-c2X polylinker (located downstream of the malE gene) is used, different vector-encoded residues are fused between the factor Xa cleavage site and the NH2-terminal methionine residue of the cloned AtCoa protein (Ile-Ser-Glu-Phe in the case of AtCoaA and Ile-Ser-Glu-Phe-Gly-Ser for AtCoaB, AtCoaD, and AtCoaE). Sequencing of the pMAL-c2X clones revealed that the sequence of AtCoaA is the same as that published in the GenBankTM. However there are three point mutations within the AtCoaD protein (A37V, I68V, and V87I) and two within the AtCoaE protein (Q186R and F204I) compared with the GenBankTM entries. The PPC synthetase activity of AtCoaB was investigated using two different malE-AtCoaB clones; in one case, the AtCoaB sequence was the same as that published in the GenBankTM, and in the other case there were five mutations (deletion of Gly45-Met46 and the point mutations D10A, M137T, R150H, and L268S). In this paper we do not differentiate between the two variants, and the term MBP-AtCoaB is used for both fusion proteins because both were active in synthesizing 4'-phosphopantothenoylcysteine. Clone AtCoaA (Ins) resulted from a nucleotide insertion in AtCoaA, giving a 36-amino acid-long inserted peptide at position 70 that replaced 15 amino acids from position 70 to position 84 of the corresponding translated protein. To improve the expression of the eukaryotic genes AtCoaB and AtCoaD, respectively, in the E. coli system, E. coli TB1 cells were co-transformed with pMal-C2XAtCoaB/AtCoaD and the helper plasmid pSBETa. The positive co-transformed colonies were selected on 200 µg/ml ampicillin and 100 µg/ml kanamycin.

Purification of Proteins
Growth of Strains—The E. coli strains used were grown to A578 = 0.4 in 0.5 liters of B-broth (10 g of casein hydrolysate 140; Invitrogen), 5 g of yeast extract (Difco), 5 g of NaCl, 1 g of glucose, and 1 g of K2HPO4 per liter, pH 7.3) in 2-liter shaker flasks, induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside (IPTG), and harvested 2 h after induction. E. coli TB1 (pMal-c2X AtCoaA/AtCoaE) cells were grown in the presence of 200 µg/ml ampicillin, E. coli TB1 (pMal-c2X AtCoaB/AtCoaD, pSBETa) cells were grown in the presence of 200 µg/ml ampicillin and 100 µg/ml kanamycin, E. coli BL21 (DE3) pET28a(+) AtHAL3a cells were grown in the presence of 100 µg/ml kanamycin, and E. coli M15 (pQE8 coaA/coaB, pREP4) cells were grown in the presence of 100 µg/ml ampicillin and 25 µg/ml kanamycin. Growth temperature was 37 °C.

MBP Fusion Proteins—For purification of the MBP-AtCoaA/B/D/E fusion proteins, IPTG-induced cells were harvested, the pellet was resuspended in 20 mM Tris-HCl (pH 8.0) buffer, and the cells were disrupted by sonication. A cleared lysate obtained by two centrifugation steps (each for 20 min at 30,000 x g at 4 °C) was adjusted to 250 mM NaCl and then applied to amylose resin equilibrated with column buffer (20 mM Tris-HCl (pH 8.0) and 250 mM NaCl). The resin was then washed with 7 volumes of column buffer. Amylose-bound proteins were eluted with 10 mM maltose in column buffer.

Purification of His-AtHAL3a Proteins—500 ml of IPTG-induced E. coli BL21 (DE3) pET28a(+) AtHAL3a cells were harvested and disrupted by sonication in 10 ml of 20 mM Tris-HCl (pH 8.0). 2.5 ml of the cleared lysate obtained by two centrifugation steps (each for 20 min at 30,000 x g at 4 °C) was diluted with a 2.5-ml column buffer (20 mM Tris-HCl (pH 8.0), 10 mM imidazole, and 300 mM NaCl) and applied to an equilibrated Ni-NTA column containing ~0.5 ml of Ni-NTA agarose (Qiagen). The column was then washed with a 10-ml column buffer. His-AtHAL3a was eluted with a column buffer containing 250 instead of 10 mM imidazole.

Purification of E. coli His-CoaA and His-CoaB Proteins—For purification of E. coli His-CoaA and His-CoaB proteins, 500 ml of IPTG-induced E. coli M15 (pREP4, pQE8 coaA/coaB) cells were harvested and disrupted by sonication in 10 ml of 20 mM Tris-HCl (pH 8.0). 1.3–2.6 ml of the cleared lysates obtained by two centrifugation steps (each for 20 min at 30,000 x g at 4 °C) were applied to Ni-NTA spin columns (Qiagen) equilibrated with column buffer (20 mM Tris-HCl (pH 8.0), 10 mM imidazole, and 300 mM NaCl). The spin columns were then washed twice with 0.65 ml of column buffer. His-CoaA and His-CoaB proteins were eluted with 0.16–0.6 ml of column buffer containing 250 instead of 10 mM imidazole. The Ni-NTA spin columns were centrifuged at room temperature at only 240 x g to enable effective binding of the His tag proteins.

SDS-PAGE—Proteins were separated using tricine-sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis under reducing conditions (28). Prestained molecular weight standards were obtained from New England Biolabs.

Activity Assays
PPC Synthetase Assay—Because 4'-phosphopantothenate is not commercially available, it was synthesized enzymatically by adding E. coli His-CoaA, pantothenate, and ATP to the PPC synthetase assay mixtures (29). Therefore, 1-ml assay mixtures contained 5 mM pantothenate, 2.5 mM MgCl2, 5–10 mM ATP (and, in addition, 5 mM CTP for E. coli His-CoaB), 5 mM cysteine hydrochloride, 10 mM DTT, 100 mM Tris (pH 8.0), His-CoaA (~15 to 25 µg), and either wild type E. coli His-CoaB or MBP-AtCoaB proteins in the range of 5–30 µg. After 45 min of incubation at 37 °C, the reaction mixtures were kept at –80 °C and then were successively separated by reversed phase chromatography (RPC) with a µRPC C2/C18 SC 2.1/10 column on a SMART system (Pharmacia). Compounds were eluted with a linear gradient of 0–50% acetonitrile-0.1% trifluoroacetic acid in 5.8 ml with a flow rate of 200 µl/min. The absorbance was measured simultaneously at 214, 260, and 280 nm to enable identification of acyl-cytidylate and acyl-adenylate intermediates, respectively (29).

4'-Phosphopantetheine Adenylyltransferase and Dephospho-CoA Kinase Assay and Reconstitution of Plant Coenzyme A Biosynthesis— 0.9-ml assay mixtures containing 5 mM pantothenate, 15 mM ATP, 15 mM MgCl2, 5 mM cysteine hydrochloride, 10 mM DTT, and 100 mM Tris (pH 8.0) were incubated with E. coli His-CoaA or MBP-AtCoaA and different combinations of the enzymes MBP-AtCoaB, His-AtHAL3a, MBP-AtCoaD, and MBP-AtCoaE (~25–50 µg each) for1hat37 °C. The reaction mixtures were then kept at –80 °C and separated by reversed phase chromatography as described above. As standard substances, synthetic dephospho-coenzyme A (Sigma) and coenzyme A (Fluka) were used.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Isolation of A. thaliana CoA Biosynthetic Genes—The complete sequence of the Arabidopsis genome (22) has been used for identification of the four remaining uncharacterized genes of the A. thaliana CoA biosynthetic pathway. With the known bacterial and human coenzyme A biosynthetic genes/proteins and using the program BLAST and the ProDom tool, five putative monofunctional A. thaliana CoA biosynthetic genes, named AtCoaA, AtCoaB, AtCoaB*, AtCoaD, and AtCoaE were identified in the comparative genomic approach. Together with the flavoprotein AtHAL3a (AtCoaC1) (gi 5802225), which was recently characterized as 4'-phosphopantothenoylcysteine decarboxylase (17), and AtHAL3b (AtCoaC2) (gi 2047324), the encoded proteins reconstitute the complete Arabidopsis CoA biosynthetic pathway (Fig. 1).

AtCoaA—A conceptual translated pantothenate kinase-related protein (gi 30696417), similar to the pantothenate kinase (gi 4191500) from Aspergillus nidulans (30), was detected as a locus product of A. thaliana chromosome 1. The A. thaliana AtCoaA protein (383 amino acids) shows only poor homology to the pantothenate kinase (gi 145561) from Escherichia coli (10). However, there is strong homology to pantothenate kinases from lower and higher eukaryotes, namely the putative pantothenate kinase (gi 6320740) encoded on chromosome IV of Saccharomyces cerevisiae and the Homo sapiens pantothenate kinase 1 isoform {beta} (gi 23510402) on chromosome 10 (31) (Fig. 2). AtCoaA (gi 30696417) also has homology to the aminoterminal domain of a further putative A. thaliana protein encoded on chromosome 4 (gi 7270122) and to the amino-terminal domain of pantothenate kinase 2 from H. sapiens (gi 20043247). The latter is encoded by the panK2 gene on chromosome 1, which is defective in Hallervorden-Spatz syndrome (32). The AtCoaA gene shows also low homology to further hypothetical bacterial representatives (for example Bacillus cereus, gi 29896568, and Bacillus anthracis, gi 21400792).



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 2.
AtCoaA alignment with homologous proteins. The predicted amino acid sequence of A. thaliana AtCoaA was compared with homologous representatives from H. sapiens and S. cerevisiae. Residues are in black boxes if two of three residues at a position are identical. Conserved residues are in gray boxes. Only the part of the comparison emphasizing the conserved residues is shown.

 

AtCoaB and AtCoaB*—Two A. thaliana proteins, AtCoaB (317 amino acids; gi 26451224, chromosome 1) and AtCoaB* (270 amino acids; gi 7340682, chromosome 5), which share 90% sequence identity, were identified as homologues of the H. sapiens monofunctional phosphopantothenoylcysteine synthetase (gi 10433192) (12). There are two significant differences between the GenBankTM entries for AtCoaB and AtCoaB*. In AtCoaB, a 24-amino acid amino-terminal extension (Met1 to Asn24) and an insertion of 22 amino acid residues (Val127 to Glu148) are present that are missing in AtCoaB*. AtCoaB and AtCoaB* both share a strong similarity, not only with the above mentioned human ortholog but also with an uncharacterized protein from yeast (gi 577131 of chromosome IX). Both the human (12) and the plant PPC synthetase show only very little sequence similarity with the E. coli enzyme (the COOH-terminal CoaB domain of the bifunctional Dfp (CoaBC) protein); however, several residues that were shown to be important for activity and/or structure of the E. coli enzyme (29) are conserved in the eukaryotic enzymes (Fig. 3A).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 3.
AtCoaB and AtCoaC alignments with homologous proteins. A, comparison of predicted AtCoaB with human and yeast counterparts. Underlined motifs are important for dimerization and/or activity of the 4'-phosphopantothenoylcysteine synthetase domain of the bifunctional E. coli Dfp flavoprotein (CoaBC). The asterisk marks the strictly conserved Lys289 residue, which is essential for the Dfp CoaB domain activity (29). B, alignment of AtCoaC1 (AtHAL3a) with the human and yeast monofunctional 4'-phosphopantothenoylcysteine decarboxylases and the Dfp CoaC domain. The conserved substrate binding clamp is underlined, and the strictly conserved residues His90 and Cys175 (of AtCoaC1) are labeled with asterisks.

 

AtCoaC1 and AtCoaC2—The flavoproteins AtHAL3a (AtCoaC1, 209 amino acids) and AtHAL3b (AtCoaC2, 201 amino acids) were first characterized as homologues of the yeast halotolerance protein SIS2/HAL3 (33–35). Later, it was shown that AtHAL3a shares the PPC decarboxylase signature sequence with bacterial enzymes (15) and indeed catalyzes the decarboxylation of PPC (Ref. 17; the PPC decarboxylase activity of AtHAL3b has not been experimentally investigated). Furthermore, the oxidatively decarboxylated intermediate of the decarboxylation reaction was purified (16), and AtHAL3a was co-crystallized with this intermediate (18). The PPC decarboxylase signature sequence is present in the H. sapiens ortholog (gi 15680133) (12) but not in SIS2. For example, the conserved Asn residue, which is involved in binding the carboxylate group of PPC, or the conserved cysteine residue of the substrate binding clamp, which is involved in reduction of the oxidatively decarboxylated intermediate, are not present in SIS2 (15, 17, 18). Therefore, it is unlikely that the yeast protein SIS2 also has PPC decarboxylase activity, and its enzymatic function remains unidentified, although sequence comparison indicate that SIS2 has a flavin binding motif (8, 36). Two further homologues of AtHAL3a are present in the Saccharomyces genome, ORF YOR054c (gi 1420190, chromosome XV) and ORF YKL088w (gi 486131, chromosome XI). Interestingly, ORF YKL088w shares the mentioned signature sequence with bacterial PPC decarboxylases and is therefore the most serious candidate for the ascription of a PPC decarboxylase activity in yeast CoA biosynthesis (Fig. 3B).

AtCoaD—The A. thaliana hypothetical protein product (gi 4309741; 176 amino acids, chromosome 2) was identified as a homologue of the 4'-phosphopantetheine adenylyltransferase domain of the H. sapiens bifunctional enzyme 4'-phosphopantetheine adenylyl transferase/dephospho-coenzyme A kinase (gi 17981025; CoaDE) from chromosome 17 (12, 37) (Fig. 4A). Although strong homology was also observed with other bifunctional eukaryotic enzymes including Mus musculus (gi 21780289) (38), Drosophila melanogaster chromosome 3L gene product (gi 10728128), and yeast chromosome VII putative monofunctional enzyme ORF YGR277c (gi 1323505), the shared sequence homology with the bacterial E. coli monofunctional adenylyltransferase (gi 125331) (7) was low.



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 4.
AtCoaD and AtCoaE alignment with homologous proteins. A, homology of the predicted AtCoaD with the putative yeast monofunctional 4'-phosphopantetheine adenylyltransferase and the CoaD domain of bifunctional human CoaDE. B, alignment of the predicted AtCoaE enzyme with human, yeast, and E. coli monofunctional relatives and the CoaE domain of bifunctional human CoaDE. The characterized Walker kinase motif GGIGSGKST is located between residues 9 and 17 in E. coli CoaE.

 

AtCoaE—Homologous representatives of the putative AtCoaE protein (232 amino acids) from A. thaliana (gi 4314391, chromosome 2) were found in bacteria, metazoa, fungi, and plants but not in archaea. There is high homology to an Oryza sativa chromosome 1 hypothetical protein (gi 14587322) and, at a lower level, to the putative CoaE enzymes of human (gi 17390295), house mouse (gi 26341674), fruit fly (gi 20151879), and bakers' yeast (gi 755789) and the recently identified E. coli monofunctional dephospho-coenzyme A kinase (9). Surprisingly, the lowest identity is observed for the dephospho-coenzyme A kinase domain of the bifunctional human CoaDE enzyme (12). The Walker kinase/ATP binding motif (27) 8GGIASGKST16 of the A. thaliana enzyme is well conserved in the other eukaryotic enzymes and in E. coli CoaE; however, there are several amino acid exchanges in the bifunctional human CoaDE enzyme that could be related to different binding constants for ATP and/or different activity rates (Fig. 4B).

Expression and Purification of Coenzyme A Biosynthetic Enzymes—The putative AtCoaA, AtCoaB, AtCoaD, and AtCoaE genes were expressed as MBP fusion proteins and purified from the corresponding E. coli clones by amylose affinity chromatography, whereas AtHAL3a and the E. coli coaB (3'-terminal part of the dfp gene) genes were expressed as His-tagged fusion proteins and purified by immobilized metal affinity chromatography (IMAC; Fig. 5).



View larger version (82K):
[in this window]
[in a new window]
  		FIG. 5.
Purification of AtCoa proteins. Purification of MBP fusion proteins (MBP-AtCoaA, MBP-AtCoaA (Ins) (not shown), MBPAtCoaB, MBP-AtCoaD, and MBP-AtCoaE) by amylose affinity chromatography and His-tagged fusion proteins (His-AtHAL3a and His-CoaA (E. coli; not shown)) by immobilized metal affinity chromatography was followed by SDS-PAGE; M, prestained molecular weight marker. Purified MBP and His-tagged fusion proteins are indicated by asterisks.

 

Identification of the PPC-Synthetase Activity of A. thaliana— The two-step conversion of 4'-phosphopantothenate to 4'-phosphopantetheine (peptide bond formation between 4-phosphopantothenate and cysteine and then decarboxylation of the formed PPC) is the key reaction in coenzyme A biosynthesis, because the reactive cysteamine residue of coenzyme A is introduced in this way. In bacteria, the formation of 4'-phosphopantetheine is catalyzed by the bifunctional Dfp flavoprotein (8, 11, 15, 29). The carboxyl-terminal CoaB domain of Dfp catalyzes the synthesis of PPC from 4'-phosphopantothenate and cysteine in two half-reactions via the acyl-cytidylate intermediate 4'-phosphopantothenoyl-CMP (29). The amino-terminal CoaC domain then catalyzes the decarboxylation of PPC using FMN as cofactor by an initial oxidation reaction (8, 16, 39). To identify the PPC synthetase activity of A. thaliana, the fusion protein MBP-AtCoaB was incubated with 4'-phosphopantothenate and cysteine in presence of the coenzymes ATP and CTP; the enzymatic activity was analyzed using the described HPLC-based assay (Fig. 6). It turned out that MBPAtCoaB is active in synthesizing PPC using ATP as cofactor. We propose that in A. thaliana the synthesis of PPC occurs via 4'-phosphopantothenoyl-AMP, because careful analysis of the HPLC data of the assays containing no cysteine reveals a compound that has a higher absorbance at 260 nm than at 280 nm. This compound is not present when the E. coli enzyme His-CoaB is used (data not shown; the intermediate of the E. coli enzyme, 4'-phosphopantothenoyl-CMP, has a higher absorbance at 280 nm than at 260 nm). Recently, it was shown that the cloned human PPC synthetase also uses ATP as cofactor, and the activity of this enzyme was determined by measuring the release of pyrophosphate (12). This supports our view that 4'-phosphopantothenoyl-AMP is the intermediate in synthesis of PPC in eukaryotes, whereas 4'-phosphopantothenoyl-CMP is the intermediate in prokaryotes (5, 11, 29). However, Abiko et al. showed that the PPC synthetase from rat liver converts ATP to ADP and phosphate (40), indicating that not all eukaryotic enzymes use the same substrate activation mechanism. Further studies are necessary to elucidate the ATP binding site of the eukaryotic enzymes and the CTP binding site of the prokaryotic counterparts. It remains open whether AtCoaB* also has PPC synthetase activity.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 6.
The plant PPC-synthetase is an ATP dependent enzyme. The synthesis of 4'-phosphopantothenoylcysteine from 4'-phosphopantothenate and cysteine was analyzed using the described HPLC-based assay in the presence of ATP omitting cysteine (A), the presence of ATP and CTP omitting cysteine (B), the presence of ATP and cysteine (C), and the presence of ATP, CTP, and cysteine (D). The absorbance was measuredsimultaneously at 214, 260 (not shown), and 280 nm (not shown). 4'-phosphopantothenate was synthesized in situ by incubation of pantothenate with ATP, Mg2+, and E. coli pantothenate kinase (His-CoaA). As a positive control, the E. coli His-CoaB enzyme was used (+ His-CoaB); in the negative control only pantothenate kinase is present (– CoaB). The putative A. thaliana PPC synthetase AtCoaB was used as MBP fusion protein (+ MBP-AtCoaB). In contrast to E. coli His-CoaB, MBP-AtCoaB does not need the cofactor CTP but synthesizes PPC in the presence of ATP and cysteine. E. coli His-CoaB synthesizes PPC via the 4'-phosphopantothenoyl-CMP intermediate (I), whereas the putative intermediate of MBPAtCoaB is 4'-phosphopantothenoyl-AMP.

 

Phosphorylation of pantothenate is essential for AtCoaB activity, because MBP-AtCoaB is not able to synthesize pantothenoylcysteine from pantothenate and cysteine (data not shown). This verifies that, in plants also, coenzyme A is synthesized from phosphorylated precursor molecules.

The Conversion of 4'-Phosphopantothenate to Coenzyme A Is Catalyzed in Four Steps by AtCoaB, AtHAL3a, AtCoaD, and AtCoaE—To analyze the activity of the putative AtCoa enzymes, 4'-phosphopantothenate was generated using the E. coli pantothenate kinase, because in these first experiments it was unclear whether AtCoaA is active. By adding different combinations of the enzymes MBP-AtCoaB, His-AtHAL3a (His-AtCoaC), MBP-AtCoaD, and MBP-AtCoaE to a reaction mixture containing pantothenate, ATP, Mg2+, cysteine, DTT, and E. coli His-CoaA, the compounds PPC, 4'-phosphopantetheine, dephospho-CoA, and coenzyme A were synthesized in vitro (Fig. 7). This clearly demonstrates that MBP-AtCoaD has 4'-phosphopantetheine adenylyltransferase and that MBP-AtCoaE has dephospho-CoA kinase activity. Dephospho-CoA cannot be completely converted to CoA, because the used molar ration of ATP to pantothenate was only 3:1. AtCoaD is specific for 4'-phosphopantetheine and does not accept 4'-phosphopantothenoylcysteine as a substrate (Fig. 8B), as also has been shown for the 4'-phosphopantetheine adenylyltransferase activity from rat liver (41).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 7.
The conversion of 4'-phosphopantothenate to coenzyme A by plant enzymes. The activity of the enzymes MBP-AtCoaB, His-AtHAL3a, MBP-AtCoaD, and MBP-AtCoaE was determined by adding combinations of the enzymes (A, MBP-AtCoaB; B, MBP-AtCoaB and His-AtHAL3a; C, MBP-AtCoaB, His-AtHAL3a and MBP-AtCoaD; D, MBP-AtCoaB, His-AtHAL3a, MBP-AtCoaD, and MBP-AtCoaE) to a reaction mixture containing pantothenate, ATP, Mg2+, cysteine, DTT, and His-CoaA (E. coli), separating the reaction products by RPC, and monitoring the absorbance at 214, 260, and 280 nm (not shown). Synthetic dephospho-CoA (E) and CoA (F) as standard substances were incubated under identical conditions without adding the plant enzymes. The high absorbances of synthesized dephospho-CoA and CoA are not in the linear range of the µPeak monitor (Amersham Biosciences) used any longer. 4'-phosphopantetheine, was identified by its slightly lower retention time compared with PPC (8).

 


View larger version (28K):
[in this window]
[in a new window]
  		FIG. 8.
In vitro reconstitution of plant coenzyme A biosynthesis and the specificity of the phosphopantetheine adenylyltransferase AtCoaD. For in vitro reconstitution of plant coenzyme A biosynthesis, MBP-AtCoaA, MBP-AtCoaB, His-AtHAL3a, MBP-AtCoaD, and MBP-AtCoaE were added to a reaction mixture containing pantothenate, ATP, Mg2+, cysteine, and DTT (C). Using RPC, the reaction products were identified to be CoA and dephospho-CoA in comparison with standard substances; the absorbance was measured simultaneously at 214, 260, and 280 nm (not shown). If His-AtHAL3a (His-AtCoaC) was omitted in the assay (B), only minor amounts of compounds with high absorbances at 260 and 280 nm were identified (labeled with an asterisk; these substances are probably {alpha}-carboxy coenzyme A and dephospho-{alpha}-carboxy-coenzyme A), indicating that AtCoaD is specific for 4'-phosphopantetheine and does not accept 4'-phosphopantothenoylcysteine, PPC, as a substrate. In a control experiment (A) it was shown that PPC is synthesized in the presence of MBP-AtCoaA and MBP-AtCoaB.

 

In Vitro Reconstitution of Plant Coenzyme A Biosynthesis— After we had verified that 4'-phosphopantothenate can be converted to coenzyme A using the enzymes MBP-AtCoaB, His-AtHAL3a (His-AtCoaC), MBP-AtCoaD, and MBP-AtCoaE, the activity of the putative pantothenate kinase MBP-AtCoaA was analyzed (Fig. 8). From the experiments that were conducted we then could conclude that MBP-AtCoaA has indeed pantothenate kinase activity, because PPC is synthesized from pantothenate and cysteine in the presence of MBP-AtCoaA and MBP-AtCoaB, and coenzyme A and dephospho-coenzyme A are synthesized if, additionally, MBP-AtHAL3a, MBP-AtCoaD, and MBP-AtCoaE are present. If MBP-AtCoaA (Ins) is used instead of MBP-AtCoaA, no pantothenate kinase activity was observed, showing that the 36-amino acid insertion into AtCoaA at position 70 inactivates the enzyme. The in vitro reconstitution of the entire biosynthetic pathway confirms that the correct A. thaliana genes have been cloned and expressed.

Conclusions—The entire biosynthetic pathway has now been elucidated in three organisms, i.e. E. coli, human, and A. thaliana. The identification of all genes necessary for biosynthesis of coenzyme A from pantothenate enables further comparative biochemical and structural studies of the involved enzymes. The existence of bifunctional CoaBC and CoaDE homologues in nature strongly indicates that AtCoaB and AtCoaC and, on the other hand, AtCoaD and AtCoaE interact with each other (42). Cloning of all plant genes will also help to elucidate the in vivo function of AtHAL3a (AtCoaC) in salt and osmotic tolerance and growth.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Research Grant KU869/6-1 and research fellowship KU 869/9-1 (to T. K.) and Spanish Ministry of Science and Technology (MCT) Research Grant BMC2002-03128 (to P. H.-A. and F. A. C.-M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence may be addressed. E-mail: thomas.kupke{at}t-online.de.

|| To whom correspondence may be addressed. E-mail: faculia{at}ibmcp.upv.es.

1 The abbreviations used are: PPC, (R)-4'-phospho-N-pantothenoylcysteine; AtCoaA/B/C/D/E, A. thaliana coenzyme A biosynthetic enzymes A/B/C/D/E; DTT, dithiothreitol; gi, gene identification number; HPLC, high pressure liquid chromatography; His-CoaA, MRGSHHHHHHGSML-CoaA; His-CoaB, MRGSHHHHHHG-Dfp Ser181 to Arg406; IPTG, isopropyl-{beta}-D-thiogalactopyranoside; MBP, maltose-binding protein from Escherichia coli; Ni-NTA, nickel nitrilotriacetic acid; ORF, open reading frame; RPC, reversed phase chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Regine Stemmler for excellent technical assistance, Professors Florence Vignols and Yves Meyer from University of Perpignan for advice and providing us with the pSBETa helper vector, and Alexis González-Policarpo for computer software help.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Abiko, Y. (1975) in Metabolic Pathways (Greenburg, D. M., ed) pp. 1–25, Academic Press, Inc., New York
  2. Kleinkauf, H. (2000) Biofactors 11, 91–92[Medline] [Order article via Infotrieve]
  3. Lynen, F. (1973) Nobel Lectures: Physiology or Medicine, 1963–1970, pp. 103–138, American Elsevier, New York
  4. Begley, T. P., Kinsland, C., and Strauss, E. (2001) Vitam. Horm. 61, 157–171[Medline] [Order article via Infotrieve]
  5. Brown, G. M. (1959) J. Biol. Chem. 234, 370–378[Free Full Text]
  6. Jackowski, S. (1996) in Escherichia coli and Salmonella, Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, pp. 687–694, ASM Press, Washington, D. C.
  7. Geerlof, A., Lewendon, A., and Shaw, W. V. (1999) J. Biol. Chem. 274, 27105–27111[Abstract/Free Full Text]
  8. Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838–31846[Abstract/Free Full Text]
  9. Mishra, P., Park, P. K., and Drueckhammer, D. G. (2001) J. Bacteriol. 183, 2774–2778[Abstract/Free Full Text]
  10. Song, W. J., and Jackowski, S. (1992) J. Bacteriol. 174, 6411–6417[Abstract/Free Full Text]
  11. Strauss, E., Kinsland, C., Ge, Y., McLafferty, F. W., and Begley, T. P. (2001) J. Biol. Chem. 276, 13513–13516[Abstract/Free Full Text]
  12. Daugherty, M., Polanuyer, B., Farrell, M., Scholle, M., Lykidis, A., De Crécy-Lagard, V., and Osterman, A. (2002) J. Biol. Chem. 277, 21431–21439[Abstract/Free Full Text]
  13. Heldt, H. W. (1997) in Plant Biochemistry & Molecular Biology (Heldt., H. W., ed) pp. 124–128, Oxford University Press Inc., New York
  14. Falk, K. L., and Guerra, D. J. (1993) Arch. Biochem. Biophys. 301, 424–430[CrossRef][Medline] [Order article via Infotrieve]
  15. Kupke, T. (2001) J. Biol. Chem. 276, 27597–27604[Abstract/Free Full Text]
  16. Hernández-Acosta, P., Schmid, D. G., Jung, G., Culiáñez-Macià, F. A., and Kupke, T. (2002) J. Biol. Chem. 277, 20490–20498[Abstract/Free Full Text]
  17. Kupke, T., Hernández-Acosta, P., Steinbacher, S., and Culiáñez-Macià, F. A. (2001) J. Biol. Chem. 276, 19190–19196[Abstract/Free Full Text]
  18. Steinbacher, S., Hernández-Acosta, P., Bieseler, B., Blaesse, M., Huber, R., Culiáñez-Macià, F. A., and Kupke, T. (2003) J. Mol. Biol. 327, 193–202[CrossRef][Medline] [Order article via Infotrieve]
  19. Gerdes, S. Y., Scholle, M. D., D'Souza, M., Bernal, A., Baev, M. V., Farrell, M., Kurnasov, O. V., Daugherty, M. D., Mseeh, F., Polanuyer, B. M., Campbell, J. W., Anantha, S., Shatalin, K. Y., Chowdhury, S. A., Fonstein, M. Y., and Osterman, A. L. (2002) J. Bacteriol. 184, 4555–4572[Abstract/Free Full Text]
  20. Choudhry, A. E., Mandichak, T. L., Broskey, J. P., Egolf, R. W., Kinsland, C., Begley, T. P., Seefeld, M. A., Ku, T. W., Brown, J. R., Zalacain, M., and Ratnam, K. (2003) Antimicrob. Agents Chemother. 47, 2051–2055[Abstract/Free Full Text]
  21. Zhao, L., Allanson, N. M., Thomson, S. P., Maclean, J. K., Barker, J. J., Primrose, W. U., Tyler, P. D., and Lewendon, A. (2003) Eur. J. Med. Chem. 38, 345–349[CrossRef][Medline] [Order article via Infotrieve]
  22. The Arabidopsis Genome Initiative (2000) Nature 408, 796–815[CrossRef][Medline] [Order article via Infotrieve]
  23. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410[CrossRef][Medline] [Order article via Infotrieve]
  24. Schenk, P. M., Baumann, S., Mattes, R., and Steinbiss, H.-H. (1995) Biotechniques 19, 196–200[Medline] [Order article via Infotrieve]
  25. Corpet, F., Servant, F., Gouzy, J., and Kahn, D. (2000) Nucleic Acids Res. 28, 267–269[Abstract/Free Full Text]
  26. Traut, T. W. (1994) Eur. J. Biochem. 222, 9–19[Medline] [Order article via Infotrieve]
  27. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945–951[Medline] [Order article via Infotrieve]
  28. Schägger, H., and Jagow, G. (1987) Anal. Biochem. 166, 368–379[CrossRef][Medline] [Order article via Infotrieve]
  29. Kupke, T. (2002) J. Biol. Chem. 277, 36137–36145[Abstract/Free Full Text]
  30. Calder, R. B., Williams, R. S., Ramaswamy, G., Rock, C. O., Campbell, E., Unkles, S. E., Kinghorn, J. R., and Jackowski, S. (1999) J. Biol. Chem. 274, 2014–2020[Abstract/Free Full Text]
  31. Ni, X., Ma, Y., Cheng, H., Jiang, M., Ying, K., Xie, Y., and Mao, Y. (2002) Int. J. Biochem. Cell Biol. 34, 109–115[CrossRef][Medline] [Order article via Infotrieve]
  32. Zhou, B., Westaway, S. K., Levinson, B., Johnson, M. A., Gitschier, J., and Hayflick, S. J. (2001) Nat. Genet. 28, 345–349[CrossRef][Medline] [Order article via Infotrieve]
  33. Espinosa-Ruiz, A., Bellés, J. M., Serrano, R., and Culiáñez-Macià, F. A. (1999) Plant J. 20, 529–539[CrossRef][Medline] [Order article via Infotrieve]
  34. Di Como, C. J., Bose, R., and Arndt, K. T. (1995) Genetics 139, 95–107[Abstract]
  35. Ferrando, A., Kron, S. J., Rios, G., Fink, G. R., and Serrano, R. (1995) Mol. Cell. Biol. 15, 5470–5481[Abstract]
  36. Blaesse, M., Kupke, T., Huber, R., and Steinbacher, S. (2000) EMBO J. 19, 6299–6310[CrossRef][Medline] [Order article via Infotrieve]
  37. Aghajanian, S., and Worrall, D. M. (2002) Biochem. J. 365, 13–18[Medline] [Order article via Infotrieve]
  38. Zhyvoloup, A., Nemazanyy, I., Babich, A., Panasyuk, G., Pobigailo, N., Vudmaska, M., Naidenov, V., Kukharenko, O., Palchevskii, S., Savinska, L., Ovcharenko, G., Verdier, F., Valovka, T., Fenton, T., Rebholz, H., Wang, M. L., Shepherd, P., Matsuka, G., Filonenko, V., and Gout, I. T. (2002) J. Biol. Chem. 277, 22107–22110[Abstract/Free Full Text]
  39. Strauss, E., and Begley, T. P. (2001) J. Am. Chem. Soc. 123, 6449–6450[CrossRef][Medline] [Order article via Infotrieve]
  40. Abiko, Y., Tomikawa, M., and Shimizu, M. (1968) J. Biochem. (Tokyo) 64, 115–117[Free Full Text]
  41. Abiko, Y., Suzuki, T., and Shimizu, M. (1967) J. Biochem. (Tokyo) 61, 309–312[Abstract/Free Full Text]
  42. Marcotte, E. M., Pellegrini, M., Ng, H. L., Rice, D. W., Yeates, T. O., and Eisenberg, D. (1999) Sciences 285, 751–753[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Plant Physiol.Home page
S. Rubio, L. Whitehead, T. R. Larson, I. A. Graham, and P. L. Rodriguez
The Coenzyme A Biosynthetic Enzyme Phosphopantetheine Adenylyltransferase Plays a Crucial Role in Plant Growth, Salt/Osmotic Stress Resistance, and Seed Lipid Storage
Plant Physiology, September 1, 2008; 148(1): 546 - 556.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
F. Bosveld, A. Rana, P. E. van der Wouden, W. Lemstra, M. Ritsema, H. H. Kampinga, and O. C. M. Sibon
De novo CoA biosynthesis is required to maintain DNA integrity during development of the Drosophila nervous system
Hum. Mol. Genet., July 1, 2008; 17(13): 2058 - 2069.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Kupke and W. Schwarz
4'-Phosphopantetheine Biosynthesis in Archaea
J. Biol. Chem., March 3, 2006; 281(9): 5435 - 5444.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
S. Rubio, T. R. Larson, M. Gonzalez-Guzman, S. Alejandro, I. A. Graham, R. Serrano, and P. L. Rodriguez
An Arabidopsis Mutant Impaired in Coenzyme A Biosynthesis Is Sugar Dependent for Seedling Establishment
Plant Physiology, March 1, 2006; 140(3): 830 - 843.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
C. D. Broeckling, D. V. Huhman, M. A. Farag, J. T. Smith, G. D. May, P. Mendes, R. A. Dixon, and L. W. Sumner
Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism
J. Exp. Bot., January 1, 2005; 56(410): 323 - 336.
[Abstract] [Full Text] [PDF]

Home page
 Mol Biol EvolHome page
U. Genschel
Coenzyme A Biosynthesis: Reconstruction of the Pathway in Archaea and an Evolutionary Scenario Based on Comparative Genomics
Mol. Biol. Evol., July 1, 2004; 21(7): 1242 - 1251.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/40/38229    most recent
M306321200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kupke, T.
Right arrow Articles by Culiáñez-Macià, F. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kupke, T.
Right arrow Articles by Culiáñez-Macià, F. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati