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. 2019 Jul 23;10(4):e01146-19.
doi: 10.1128/mBio.01146-19.

Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea

Takahiro Shimosaka  1   2 , Kira S Makarova  3 , Eugene V Koonin  3 , Haruyuki Atomi  4
Affiliations

Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea

Takahiro Shimosaka et al. mBio. .

Abstract

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.

Keywords: archaea; coenzyme A; dephospho-CoA kinase; hyperthermophiles; metabolism.

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Figures

FIG 1
FIG 1
CoA biosynthesis pathways in the three domains of life. The conversion of pantoate to 4′-phosphopantothenate is catalyzed by PS and PanK in bacteria and eukaryotes. PoK and PPS replace the PS-PanK system in most archaea. Genes that encode CoA biosynthesis enzymes in T. kodakarensis are noted in parentheses. The reaction catalyzed by the novel DPCK encoded by the TK1697 gene is indicated in red, along with the genes (TK0517 and TK2128) evaluated in this study. THF, tetrahydrofolate. Other abbreviations are defined in the text.
FIG 2
FIG 2
Phosphate donor specificity of the TK1697 protein. The reaction mixture contained 1 mM dephospho-CoA, 1 mM NTP, 5 mM MgCl2, and 10 μg ml−1 recombinant protein for UTP and GTP or 50 μg ml−1 recombinant protein for ATP and CTP in 50 mM Bicine (pH 8.0).
FIG 3
FIG 3
Effect of temperature and pH on the DPCK activity of the TK1697 protein. (A) Effects of temperature on DPCK activity. (B) Arrhenius plot of the data shown in panel A. (C) Effects of pH on DPCK activity. Symbols: closed triangles, MES; open squares, HEPES; closed circles, Bicine. (D) Thermostability of the TK1697 protein. Symbols: squares, 70°C; triangles, 80°C; circles, 90°C.
FIG 4
FIG 4
Kinetic examination of the dephospho-CoA kinase reaction. DPCK activity assays were performed with various concentrations of dephospho-CoA with 5 mM GTP (A) or GTP and UTP with 1 mM dephospho-CoA (B). Symbols: closed circles, GTP; open circles, UTP. Measurements were performed at 80°C. Insets show double reciprocal plots of the data.
FIG 5
FIG 5
Growth characteristics of T. kodakarensis ΔTK1697 (A), ΔTK0517 (B), and ΔTK2128 (C) and their host strains. Cells were cultivated in ASW-YT-pyruvate-agmatine medium (A) or ASW-YT-pyruvate medium (B and C) at 85°C. Symbols: black circles, KPD1 (A) or KU216 (B and C); blue circles, disruption strains; red circles, disruption strains grown in medium supplemented with 1 mM CoA. Error bars represent the standard deviations from three independent experiments.
FIG 6
FIG 6
Growth characteristics of T. kodakarensis ΔTK1697 (ΔpyrF ΔpdaD ΔTK1697) transformed with the wild-type TK1697 gene. Cells were cultivated in ASW-YT-pyruvate medium at 85°C. Symbols: black circles, the host strain KPD1 (ΔpyrF ΔpdaD) transformed with pRPG03-f; blue circles, disruption strain transformed with pRPG03-f; red circles, disruption strain transformed with pRPETK1697. Error bars represent the standard deviations from three independent experiments.
FIG 7
FIG 7
Growth characteristics of T. kodakarensis ΔTK1697 (ΔpyrF ΔpdaD ΔTK1697) transformed with pRPETK1697(D48A), pRPETK1697(D67A), or pRPETK1697(D125A). Cells were cultivated in ASW-YT-pyruvate medium at 85°C. Symbols: black circles, disruption strain transformed with pRPETK1697; red circles, disruption strain transformed with pRPETK1697(D48A); blue circles, disruption strain transformed with pRPETK1697(D67A); green circles, disruption strain transformed with pRPETK1697(D125A). Error bars represent the standard deviations from three independent experiments.
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