Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
PLoS One. 2022 Jun 8;17(6):e0269572. doi: 10.1371/journal.pone.0269572. eCollection 2022.
Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical Escherichia coli aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. Streptococcus thermophilus RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the E. coli class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.
核糖核苷酸还原酶 (RNRs) 利用基于自由基的化学催化将所有四种核糖核苷酸转化为脱氧核糖核苷酸。RNRs 的普遍存在性需要多种 RNR 类，它们在核糖核苷酸底物的磷酸化状态、氧耐受性以及所使用的金属辅因子和还原系统的性质方面彼此不同。虽然这些差异允许 RNR 在广泛的环境条件下产生 DNA 生物合成所需的脱氧核糖核苷酸，但它们也为建立通用活性测定带来了挑战。此外，许多当前的 RNR 测定方法受到限制，因为它们一次只能跟踪一种核糖核苷酸底物的转化，但在细胞中，所有四种核糖核苷酸都在根据所有变构特异性效应物的细胞浓度积极转化为脱氧核苷酸产物。在这里，我们提出了一种基于液相色谱-串联质谱 (LC-MS/MS) 的测定方法，该方法可以使用任何变构效应物组合测定任何组合的底物上有氧和无氧 RNR 的活性。我们证明该测定方法生成的活性数据与过去发表的经典大肠杆菌有氧 Ia 类 RNR 的结果相似。我们还表明，该测定方法可用于使用甲酸作为还原剂的厌氧 III 类 RNR，即嗜热链球菌 RNR。我们进一步表明，该 III 类 RNR 受到 dATP 和 ATP 的变构调节。最后，我们展示了在各种变构特异性效应物组合下大肠杆菌 Ia 类 RNR 同时还原所有四种核糖核苷酸底物的活性数据。与历史上建立的放射性活性和偶联 RNR 活性测定以及许多已发表的 HPLC 测定方法相比，这种经过验证的 LC-MS/MS 测定方法具有更高的通量和更大的通用性。该测定方法将允许在广泛的条件下研究广泛的 RNR 酶，从而促进对以前未表征的 RNR 的研究。
