Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.
Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.
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DOI：
10.1038/srep22035
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发表时间：
2016-02-25
影响因子：
4.6
通讯作者：
Pochapsky TC
中科院分区：
文献类型：
--
作者：
Colthart AM;Tietz DR;Ni Y;Friedman JL;Dang M;Pochapsky TC
细胞色素P450单加氧酶通常催化一个氧原子从O2插入到未活化的碳-氢和碳-碳键中，同时另一个氧原子被NAD(P)H还原成H2O。通过对残留偶极偶联（RDC）约束分子动力学（MD）得到的樟脑羟化酶CYP101 （cytochrome P450cam）在存在和不存在底物时的平均结构进行比较，发现底物去除后活性位点的基本崩溃导致了结构位移。这种崩溃的构象结果延伸到整个蛋白质结构，在类似的晶体结构中没有观察到。进行突变以测试所观察到的底物结合和识别构象变化的参与。所有基于核磁共振检测扰动的突变，即使是那些远离活性位点的突变，都会导致底物选择性、酶效率和/或血红素铁自旋状态的改变。结果表明，溶液核磁共振可以提供其他方法难以获得的酶结构-功能关系的见解。
Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.