Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli *

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 fromEscherichia 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 renamedcoaD, 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.

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