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Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase from Legionella pneumophila.

ABSTRACT: Dephospho-CoA kinases (DPCKs) are members of the kinase family that catalyze the final step in CoA biosynthesis. Their function is phosphorylation of the 3-hydroxyl group of the ribose using ATP as a phosphate donor. Structural changes induced by ATP binding play an important role during the DPCK catalytic cycle. In this work, DPCK from Legionella pneumophila was overexpressed in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are described. The protein was crystallized in space group P21212, with unit-cell parameters a = 36.29, b = 82.20, c = 81.80 Å, using the hanging-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method.

SUBMITTER: Yu D

PROVIDER: S-EPMC4014329 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase from Legionella pneumophila.

Yu Dongmin D Chen Xiaofang X Xu Zhongdong Z Ge Honghua H

Acta crystallographica. Section F, Structural biology communications 20140415 Pt 5

Dephospho-CoA kinases (DPCKs) are members of the kinase family that catalyze the final step in CoA biosynthesis. Their function is phosphorylation of the 3-hydroxyl group of the ribose using ATP as a phosphate donor. Structural changes induced by ATP binding play an important role during the DPCK catalytic cycle. In this work, DPCK from Legionella pneumophila was overexpressed in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are de ...[more]

PMID: 24817720

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Project description: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 Thermococcus kodakarensis 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 T. kodakarensis, 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 T. kodakarensis 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 T. kodakarensisIMPORTANCE 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 Thermococcus kodakarensis 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.

Dataset Information

Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase from Legionella pneumophila.

Publications

Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase from Legionella pneumophila.

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