Abstract
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+.
Highlights
Coenzyme A and 4Ј-phosphopantetheine are essential cofactors for many enzymatic reactions, and acyl-CoA derivatives are key intermediates in energy metabolism [1]
4Ј-Phosphopantetheine and Coenzyme A Biosynthesis in Plants 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), which was recently characterized as 4Ј-phosphopantothenoylcysteine decarboxylase [17], and AtHAL3b (AtCoaC2), the encoded proteins reconstitute the complete Arabidopsis CoA biosynthetic pathway (Fig. 1)
Summary
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 SKϩ vector). The first strand cDNA template was PCR amplified using REDTaq DNA polymerase and the 5Ј-forward and 3Ј-reverse genespecific adapted primers 5Ј-CGAGGAATTCATGGATCCGACTCAAATCTCTC-3Ј/5Ј-CCGGCTCGAGCTAAACTAAATTGATGCTACATTC-3Ј; 5Ј-CCGGGGATCCATGAGTTCGATCTCTGGATTGGTGGAAG-3Ј/5Ј-CCGGCTCGAGTCAAGTGAGAGATTCTTTGATGTATG-3Ј; 5Ј-CCGGGGATCCATGGCAGCTCCGGAAGATTCAAAGATG-3Ј/5-CGAGAAGC TTCCACCTCATGATGCTTTTTCTTCTGCTGGTTG-3Ј; and 5Ј-CCGGGGATCCATGAGAATAGTCGGGTTAACGGG-3Ј/5Ј-CGAGAAGCTTCCACCTTAAGAGCCAATTTTGAGCTGTTTGC-3Ј for AtCoaA, AtCoaB, AtCoaD, and AtCoaE, respectively. The PCR products were cloned into the pBluescript SKϩ vector, and 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. The positive co-transformed colonies were selected on 200 g/ml ampicillin and 100 g/ml kanamycin
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