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.

• Structural basis of CTP-dependent peptide bond formation in coenzyme A biosynthesis catalyzed by Escherichia coli PPC synthetase.

  Stanitzek S., Augustin M.A., Huber R., Kupke T., Steinbacher S.

  Phosphopantothenoylcysteine (PPC) synthetase forms a peptide bond between 4'-phosphopantothenate and cysteine in coenzyme A biosynthesis. PPC synthetases fall into two classes: eukaryotic, ATP-dependent and eubacterial, CTP-dependent enzymes. We describe the first crystal structure of E. coli PPC ... >> More

  Phosphopantothenoylcysteine (PPC) synthetase forms a peptide bond between 4'-phosphopantothenate and cysteine in coenzyme A biosynthesis. PPC synthetases fall into two classes: eukaryotic, ATP-dependent and eubacterial, CTP-dependent enzymes. We describe the first crystal structure of E. coli PPC synthetase as a prototype of bacterial, CTP-dependent PPC synthetases. Structures of the apo-form and the synthetase complexed with CTP, the activated acyl-intermediate, 4'-phosphopantothenoyl-CMP, and with the reaction product CMP provide snapshots along the reaction pathway and detailed insight into substrate binding and the reaction mechanism of peptide bond formation. Binding of the phosphopantothenate moiety of the acyl-intermediate in a cleft at the C-terminal end of the central beta sheet of the dinucleotide binding fold is accomplished by an otherwise flexible flap. A second disordered loop may control access of cysteine to the active site. The conservation of functionalities involved in substrate binding and catalysis provides insight into similarities and differences of prokaryotic and eukaryotic PPC synthetases. << Less

  Structure 12:1977-1988(2004) [PubMed] [EuropePMC]