Krieger Célia, Roselli Sandro, Kellner-Thielmann Sandra, Galati Gianni, Schneider Bernd, Grosjean Jérémy, Olry Alexandre, Ritchie David, Matern Ulrich, Bourgaud Frédéric, Hehn Alain
Laboratoire Agronomie et Environnement, Institut National de la Recherche Agronomique, Université de Lorraine, Nancy, France.
Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Marburg, Germany.
Front Plant Sci. 2018 Jun 19;9:820. doi: 10.3389/fpls.2018.00820. eCollection 2018.
The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from and CYP71AZ3, 4, and 6 from were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.
高等植物中香豆素和呋喃香豆素（FCs）的产生被广泛认为是植物适应环境的一个典型例证。在本报告中，我们表明CYP71AZ亚科内细胞色素P450变体的倍增促成了这些分子的多样化。在伞形科植物中发现了编码该酶家族的多个基因拷贝，其系统发育分析表明它们在这些植物中具有不同的功能。对来自[具体植物1]的CYP71AZ1以及来自[具体植物2]的CYP71AZ3、4和6进行了功能表征。虽然CYP71AZ3仅对七叶亭进行羟基化，但其他酶既接受简单香豆素也接受FCs。这些酶的叠加模型导致在酶活性位点鉴定出三个区域的不同构象。随后利用这些序列将CYP71AZ4突变以使其类似于CYP71AZ3。这些区域的交换导致底物特异性显著改变。所有三个区域的同时突变将CYP71AZ4的特异性转变为CYP71AZ3的特异性，仅接受七叶亭。这种方法可能解释了该细胞色素P450家族在防风草以及可能在伞形科中FCs出现方面的进化情况。
