title: Purification and characterization of phosphopantetheine adenylyltransferase from Escherichia coli.
abstract: Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4'-phosphopantetheine yielding 3'-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3'-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.
authors.affiliation: Department of Microbiology and Immunology, University of Leicester, Leicester LE1 9HN, United Kingdom. ag35@le.ac.uk
authors.forename: A
authors.initials: A
authors.lastname: Geerlof
authors.forename: A
authors.initials: A
authors.lastname: Lewendon
authors.forename: W V
authors.initials: WV
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000096:biosynthesis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C015787:dephosphocoenzyme A; D009713:Nucleotidyltransferases; C022315:pantetheine-phosphate adenylyltransferase; D003065:Coenzyme A