Publications

• Identification of dephospho-coenzyme A (dephospho-CoA) kinase in Thermococcus kodakarensis and elucidation of the entire CoA biosynthesis pathway in archaea.

  Shimosaka T., Makarova K.S., Koonin E.V., Atomi H.

  Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon <i>Thermococcus ... >> More

  Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon <i>Thermococcus kodakarensis</i> encodes two homologs of bacterial DPCK and the DPCK domain of eukaryotic CoA synthase, TK1334 and TK2192. We purified the recombinant TK1334 and TK2192 proteins and found that they lacked DPCK activity. Bioinformatic analyses showed that, in several archaea, the uncharacterized gene from arCOG04076 protein is fused with the gene for phosphopantetheine adenylyltransferase (PPAT), which catalyzes the reaction upstream of the DPCK reaction in CoA biosynthesis. This observation suggested that members of arCOG04076, both fused to PPAT and standalone, could be the missing archaeal DPCKs. We purified the recombinant TK1697 protein, a standalone member of arCOG04076 from <i>T. kodakarensis</i>, and demonstrated its GTP-dependent DPCK activity. Disruption of the TK1697 resulted in CoA auxotrophy, indicating that TK1697 encodes a DPCK that contributes to CoA biosynthesis in <i>T. kodakarensis</i> TK1697 homologs are widely distributed in archaea, suggesting that the arCOG04076 protein represents a novel family of DPCK that is not homologous to bacterial and eukaryotic DPCKs but is distantly related to bacterial and eukaryotic thiamine pyrophosphokinases. We also constructed and characterized gene disruption strains of TK0517 and TK2128, homologs of bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and PPAT, respectively. Both strains displayed CoA auxotrophy, indicating their contribution to CoA biosynthesis. Taken together with previous studies, the results experimentally validate the entire CoA biosynthesis pathway in <i>T. kodakarensis</i><b>IMPORTANCE</b> CoA is utilized in a wide range of metabolic pathways, and its biosynthesis is essential for all life. Pathways for CoA biosynthesis in bacteria and eukaryotes have been established. In archaea, however, the enzyme that catalyzes the final step in CoA biosynthesis, dephospho-CoA kinase (DPCK), had not been identified. In the present study, bioinformatic analyses identified a candidate for the DPCK in archaea, which was biochemically and genetically confirmed in the hyperthermophilic archaeon <i>Thermococcus kodakarensis</i> Genetic analyses on genes presumed to encode bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and phosphopantetheine adenylyltransferase confirmed their involvement in CoA biosynthesis. Taken together with previous studies, the results reveal the entire pathway for CoA biosynthesis in a single archaeon and provide insight into the different mechanisms of CoA biosynthesis and their distribution in nature. << Less

  MBio 10:E01146-E01146(2019) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Cubic crystals of phosphopantetheine adenylyltransferase from Escherichia coli.

  Izard T., Geerlof A., Lewendon A., Barker J.J.

  Phosphopantetheine adenylyltransferase (PPAT, E.C. 2.7.7.3) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis, transferring an adenylyl group from ATP to 4'-phosphopantetheine, and forming dephospho-CoA. Cubic crystals of native PPAT from Escherichia coli as well as PPAT in complex w ... >> More

  Phosphopantetheine adenylyltransferase (PPAT, E.C. 2.7.7.3) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis, transferring an adenylyl group from ATP to 4'-phosphopantetheine, and forming dephospho-CoA. Cubic crystals of native PPAT from Escherichia coli as well as PPAT in complex with its substrates were obtained. The crystals belong to space group I23 or I213 with unit-cell dimension a = 135.5 A. The crystals diffract to better than 1.8 A resolution on a Cu Kalpha rotating-anode generator. The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 2.9 A3 Da-1. << Less

  Acta Crystallogr D Biol Crystallogr 55:1226-1228(1999) [PubMed] [EuropePMC]

• Purification and characterization of phosphopantetheine adenylyltransferase from Escherichia coli.

  Geerlof A., Lewendon A., Shaw W.V.

  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 anal ... >> More

  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. << Less

  J. Biol. Chem. 274:27105-27111(1999) [PubMed] [EuropePMC]

• Separate enzymes catalyze the final two steps of coenzyme A biosynthesis in Brevibacterium ammoniagenes: purification of pantetheine phosphate adenylyltransferase.

  Martin D.P., Drueckhammer D.G.

  We have discovered that the final two steps in coenzyme A biosynthesis in Brevibacterium ammoniagenes are catalyzed by distinct enzymes, readily separated by DEAE sepharose anion exchange chromatography. This is in contrast to mammalian tissues in which these two reactions are catalyzed by a singl ... >> More

  We have discovered that the final two steps in coenzyme A biosynthesis in Brevibacterium ammoniagenes are catalyzed by distinct enzymes, readily separated by DEAE sepharose anion exchange chromatography. This is in contrast to mammalian tissues in which these two reactions are catalyzed by a single bifunctional enzyme (Worrall, D.M., and Tubbs, P.K. (1983) Biochem. J. 215, 153-157) and Bakers yeast in which these two activities have been identified as part of a multifunctional complex (Bucovaz, E.T., Rhoades, J.L., and Tarnowski, S.J. (1980) Fed. Proc. 39 (6), 142). The pantetheine phosphate adenylyltransferase has been purified to homogeneity and found to exist as a trimeric protein of molecular mass approximately 108 kDa. Of other nucleoside triphosphates tested as substrates, only 2'-deoxy-ATP showed measurable activity, being 27% that of ATP. << Less

  Biochem Biophys Res Commun 192:1155-1161(1993) [PubMed] [EuropePMC]

• Enzymatic synthesis and structure of CoA.

  NOVELLI G.D.

  Fed Proc 12:675-681(1953) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biosynthesis of coenzyme A from phospho-pantetheine and of pantetheine from pantothenate.

  HOAGLAND M.B., NOVELLI G.D.

  J Biol Chem 207:767-773(1954) [PubMed] [EuropePMC]