Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea
Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea
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DOI：
10.1128/jb.06624-11
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发表时间：
2012-10-01
影响因子：
3.2
通讯作者：
Atomi, Haruyuki
中科院分区：
文献类型：
--
作者：
Tomita, Hiroya;Yokooji, Yuusuke;Atomi, Haruyuki
虽然细菌和真核生物共享辅酶A（CoA）生物合成的途径，但我们先前阐明大多数古细菌利用不同的途径将泛解酸转化为4 '-磷酸泛泛酸。而细菌/真核生物使用泛酸合成酶和泛酸激酶（PanK），超嗜热古菌Thermococcus kodakarensis利用两种新的酶：泛酸激酶（PoK）和磷酸泛酸合成酶（PPS）。在这里，我们报告了一个详细的生化检查PoK从T。科达卡人。动力学分析表明，PoK反应显示Michaelis-Menten动力学对ATP，而与泛解酸观察到底物抑制。PoK活性不受CoA/乙酰辅酶A添加的影响。有趣的是，PoK显示出广泛的核苷酸特异性，并利用ATP，GTP，UTP和CTP具有可比的k（cat）/K-m值。27个PoK同源物的序列比对显示7个保守的残基与反应性侧链，并为每个残基构建变体蛋白。当突变被引入Ser 104，Glu 134和Asp 143时，没有检测到活性，这表明这些残基在PoK催化中起着至关重要的作用。其他变体蛋白，突变S28 A，H131 A，R155 A，和T186 A的动力学分析表明，所有四个残基参与泛解酸识别和Arg 155和Thr 186在PoK催化中发挥重要作用。凝胶过滤分析的变体蛋白表明，Thr 186也参与二聚体组装。PoK和GHMP激酶家族的其他成员之间的序列比较表明，Ser 104和Glu 134分别参与与磷酸盐和Mg 2+的结合，而Asp 143是负责从泛解酸羟基中提取质子的碱基。
Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k(cat)/K-m values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg2+, respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.