title: Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics.
abstract: The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.
authors.affiliation: Integrated Genomics, Incorporated, Chicago, Illinois 60612 and the Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Daugherty
authors.forename: Boris
authors.initials: B
authors.lastname: Polanuyer
authors.forename: Michael
authors.initials: M
authors.lastname: Farrell
authors.forename: Michael
authors.initials: M
authors.lastname: Scholle
authors.forename: Athanasios
authors.initials: A
authors.lastname: Lykidis
authors.forename: Valerie
authors.initials: V
authors.lastname: de Crécy-Lagard
authors.forename: Andrei
authors.initials: A
authors.lastname: Osterman
mesh_terms: D015152:Blotting, Northern; D002262:Carboxy-Lyases / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D020745:Genome, Archaeal; D016680:Genome, Bacterial; D016681:Genome, Fungal; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics; D010453:Peptide Synthases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020021:dephospho-CoA kinase; D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase; D002262:Carboxy-Lyases; C022373:Phosphopantothenoyl-cysteine decarboxylase; D010453:Peptide Synthases; C022374:phosphopantothenoyl-cysteine synthetase; D003065:Coenzyme A