Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

• Published in: The Journal of biological chemistry Vol. 274; no. 38; pp. 27105 - 27111
• Main Authors: Geerlof, Arie, Lewendon, Ann, Shaw, William V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-09-1999; American Society for Biochemistry and Molecular Biology
• Subjects: 3'-Dephospho-CoA; 4'-Phosphopantetheine; Amino Acid Sequence; Cloning, Molecular; coenzyme A; Coenzyme A - biosynthesis; Dimerization; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Models, Chemical; Molecular Sequence Data; Molecular Weight; Nucleotidyltransferases - genetics; Nucleotidyltransferases - isolation & purification; Nucleotidyltransferases - metabolism; phophopantetheine adenylyltransferase; pyrophosphate; Sequence Alignment
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.38.27105

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.