4'-phosphopantetheine and coenzyme A biosynthesis in plants.
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+.
Kupke
Thomas
T
Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany. thomas.kupke@t-online.de
Hernández-Acosta
Pilar
P
Culiáñez-Macià
Francisco A
FA
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics*; D001669:Biochemical Phenomena; D001671:Biochemistry; D002460:Cell Line; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000096:biosynthesis*; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D018745:Genome, Plant; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009995:Osmosis; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry*; D010944:Plants / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D010204:Pantetheine; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase; D008274:Magnesium; D003545:Cysteine; C003129:4'-phosphopantetheine; D003065:Coenzyme A