Abstract
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 Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico 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.
Highlights
Plants have developed a wide range of strategies to take advantage of their environment and to adapt to many conditions by methods such as the production of defense molecules
Various studies have reported the pivotal role of P450 enzymes in the synthesis of FCs (Hamerski and Matern, 1988a,b; Larbat et al, 2007, 2009)
The adaptation of plants to changed environmental conditions often requires the creation of modified or novel enzymatic capabilities, and the P450 superfamily is susceptible to such evolutionary processes as confirmed by the vast array of reactions catalyzed by this multigene family (Mizutani and Ohta, FIGURE 7 | Sequence alignment of P. sativa CYP71AZ1, CYP71AZ3, CYP71A74, and CYP71AZ6 and construction of the CYP71AZ4 mutants. (A) Alignment of the four peptide sequences
Summary
Plants have developed a wide range of strategies to take advantage of their environment and to adapt to many conditions by methods such as the production of defense molecules. This process has been studied for many decades (Fraenkel, 1959), but the molecular characterization of the enzymes responsible for the synthesis of the defense molecule arsenal primarily started in the 1990s (Funk and Croteau, 1993; Werck-Reichhart et al, 1997; Glawischnig et al, 1999). More than 70% of the 245 P450s in the Arabidopsis genome, which is smaller than many other plant genomes, are orphans at the biochemical level (Mizutani and Sato, 2012)
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