Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea
Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea
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DOI：
10.1128/mbio.01146-19
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发表时间：
2019-07-01
期刊：
影响因子：
6.4
通讯作者：
Atomi, Haruyuki
中科院分区：
文献类型：
--
作者：
Shimosaka, Takahiro;Makarova, Kira S.;Atomi, Haruyuki
脱磷酸辅酶A激酶（DPCK）催化脱磷酸辅酶A的ATP依赖性磷酸化，这是辅酶A生物合成的最后一步。DPCK已在细菌和真核生物中被鉴定和表征，但在古细菌中未被鉴定和表征。极端嗜热古菌Thermococcus kodakarensis编码细菌DPCK的两个同源物和真核生物CoA合酶的DPCK结构域TK 1334和TK 2192。我们纯化了重组TK 1334和TK 2192蛋白，发现它们缺乏DPCK活性。生物信息学分析表明，在几个古细菌中，来自arCOG 04076蛋白的未表征基因与磷酸泛酰巯基乙胺腺苷酰转移酶（PPAT）基因融合，该基因催化CoA生物合成中的DPCK反应上游的反应。这一观察结果表明，与PPAT融合和独立的arCOG 04076的成员可能是缺失的古细菌DPCK。我们从T. kodakarensis，并证明其GTP依赖性的DPCK活性。TK 1697的破坏导致辅酶A营养缺陷型，表明TK 1697编码的DPCK有助于T.科达卡人。TK 1697同源物广泛分布于古细菌中，这表明arCOG 04076蛋白代表了DPCK的一个新家族，该家族与细菌和真核生物的DPCK不同源，但与细菌和真核生物的硫胺素焦磷酸激酶有远亲关系。我们还构建了TK 0517和TK 2128的基因破坏菌株，分别是双功能磷酸泛酰半胱氨酸合成酶-磷酸泛酰半胱氨酸脱羧酶和PPAT的同源物，并对其进行了表征。这两种菌株显示CoA营养缺陷型，表明它们对CoA生物合成的贡献。结合以往的研究，实验结果验证了T.重要性辅酶A用于广泛的代谢途径，并且其生物合成对于所有生命都是必需的。已经建立了细菌和真核生物中CoA生物合成的途径。然而，在古细菌中，催化辅酶A生物合成的最后一步的酶，脱磷酸辅酶A激酶（DPCK），还没有被确定。在本研究中，生物信息学分析确定了一个候选人的DPCK在古菌，这是生物化学和遗传学证实的超嗜热古菌Thermococcus kodakarensis。对编码双功能磷酸泛酰巯基乙胺半胱氨酸合成酶-磷酸泛酰巯基乙胺半胱氨酸脱羧酶和磷酸泛酰巯基乙胺腺苷转移酶基因的遗传分析证实了它们参与辅酶A的生物合成。与以前的研究一起，结果揭示了单个古菌中CoA生物合成的整个途径，并提供了对CoA生物合成的不同机制及其在自然界中分布的见解。
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. kodakarensis.IMPORTANCE 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.