Šimelis Klemensas, Belle Roman, Kawamura Akane
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK.
Chemistry-School of Natural and Environmental Sciences, Bedson Building, Newcastle University, Newcastle upon Tyne, UK.
Commun Chem. 2024 Dec 20;7(1):305. doi: 10.1038/s42004-024-01382-1.
Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases use 2OG and O cofactors to catalyse substrate oxidation and yield oxidised product, succinate, and CO. Simultaneous detection of substrate and cofactors is difficult, contributing to a poor understanding of the dynamics between substrate oxidation and 2OG decarboxylation activities. Here, we profile 5-methylcytosine (C)-oxidising Ten-Eleven Translocation (TET) enzymes using MS and H NMR spectroscopy methods and reveal a high degree of substrate oxidation-independent 2OG turnover under a range of conditions. 2OG decarboxylase activity is substantial (>20% 2OG turned over after 1 h) in the absence of substrate, while, under substrate-saturating conditions, half of total 2OG consumption is uncoupled from substrate oxidation. 2OG kinetics are affected by substrate and non-substrate DNA oligomers, and the sequence-agnostic effects are observed in amoeboflagellate Naegleria gruberi NgTet1 and human TET2. TET inhibitors also alter uncoupled 2OG kinetics, highlighting the potential effect of 2OG dioxygenase inhibitors on the intracellular balance of 2OG/succinate.
依赖于亚铁离子（Fe(II)）和2-酮戊二酸（2OG）的双加氧酶利用2OG和氧气作为辅因子来催化底物氧化，生成氧化产物琥珀酸和二氧化碳。同时检测底物和辅因子很困难，这导致我们对底物氧化和2OG脱羧活性之间的动态关系了解不足。在这里，我们使用质谱（MS）和核磁共振氢谱（H NMR）光谱方法对5-甲基胞嘧啶（5mC）氧化的TET（Ten-Eleven Translocation）酶进行了分析，并揭示了在一系列条件下存在高度的与底物氧化无关的2OG周转。在没有底物的情况下，2OG脱羧酶活性很高（1小时后超过20%的2OG发生周转），而在底物饱和条件下，2OG总消耗量的一半与底物氧化解偶联。2OG的动力学受底物和非底物DNA寡聚物的影响，并且在变形鞭毛虫纳氏虫（Naegleria gruberi）的NgTet1和人类TET2中观察到了与序列无关的效应。TET抑制剂也会改变解偶联的2OG动力学，这突出了2OG双加氧酶抑制剂对细胞内2OG/琥珀酸平衡的潜在影响。
