A continuous fluorescence assay for the characterization of Nudix hydrolases.
A continuous fluorescence assay for the characterization of Nudix hydrolases.
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DOI：
10.1016/j.ab.2013.02.023
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发表时间：
2013-06-15
影响因子：
2.9
通讯作者：
Kirsch JF
中科院分区：
文献类型：
--
作者：
Xu A;Desai AM;Brenner SE;Kirsch JF
功能多样的NuVibration蛋白超家族的共同底物结构是核苷酸-二磷酸-X，其中X是各种各样的离去基团。在29，400个已知成员中，已知底物特异性的不到1%。大多数活性导致无机磷酸根离子或带有末端磷酸根部分的产物的释放。反应通常通过不连续Fiske-SubbaRow测定法的修改来监测，该测定法相对不敏感且缓慢。我们在这里报告的发展，使一个96孔格式的底物特异性的快速和准确的测定连续荧光检测。我们使用这种新的检测方法来证实大肠杆菌MutT和NudD的底物特征，并表征耐辐射异常球菌DR_1025和马氏甲烷八叠球菌MM_0920。新检测方法带来的新发现包括以下内容。首先，除了在α-β位置处的8-氧代-dGTP的充分表征的水解之外，MutT在β-γ磷酸键处的裂解速率为在α-β位置处的水解所记录的速率的3%。其次，MutT还催化5-甲基-dCTP的水解。第三，在筛选的41种化合物中，观察到8-氧代-dGTP是DR_1025的最佳底物。
The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske–SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α–β position, MutT cleaves at the β–γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α–β position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.
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影响因子：
2.9
作者：
Ito, R;Hayakawa, H;Ishibashi, T
通讯作者：
Ishibashi, T
影响因子：
4.1
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Ofman, R;Speijer, D;Wanders, RJA
通讯作者：
Wanders, RJA
影响因子：
2.9
作者：
Harris, TK;Wu, G;Mildvan, AS
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Mildvan, AS
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作者：
Perraud, AL;Fleig, A;Scharenberg, AM
通讯作者：
Scharenberg, AM
影响因子：
3.9
作者：
Hahn, FM;Xuan, JW;Poulter, CD
通讯作者：
Poulter, CD