The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants

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.

Substrate‐specificity of cytochrome P450‐mediated detoxification as an evolutionary strategy for specialization on furanocoumarin‐containing hostplants: CYP6AE89 in parsnip webworms

“…The furanocoumarins have long been regarded as an example of evolution of plant chemical defence in an escalating fashion. The umbelliferone precursor of these compounds is widely distributed across plant taxa, whereas the linear furanocoumarins are restricted to fewer families, and angular furanocoumarins are even more limited in distribution (Berenbaum, 1983;Krieger et al, 2018). CYP6AE89, which may have evolved prior to the evolution of several other webworm CYP6AE enzymes, is specific for linear furanocoumarins but does not metabolize the angular furanocoumarins in Pas.…”

Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants

“…The UHPLC‐PDA analysis was performed essentially as described by Krieger et al . (). In the prenyl donor specificity test, acetonitrile was used instead of methanol as a solvent.…”

“…Cytochrome P450 monooxygenases (P450s) were described as being responsible for furan‐ring formation; genes encoding these enzymes include CYP71AJ1 , which encodes a psoralen synthase (PS) (Larbat et al ., ), and CYP71AJ4 , which encodes an angelicin synthase (AS) (Larbat et al ., ). Other P450 genes involved in subsequent modifications of the FC cores include CYP71AZ4 , which encodes a psoralen 8‐hydroxylase (Krieger et al ., ), and CYP71AZ1/6 , which encodes a xanthotoxin 5‐hydroxylase (Krieger et al ., ). Bergaptol 5‐ O ‐methyltransferases have also been reported to belong to the SABATH superfamily (Hehmann et al ., ; Ishikawa et al ., ; Zhao et al ., ) (Fig.…”

A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris

“…The PCR product was ligated into the TA cloning vector pCR™8/GW/ TOPO™ system (Invitrogen™, Thermo Fisher Scientific) and subsequently sequenced. The gene was further cloned by LR recombination using LR Clonase II™ (Invitrogen™, Thermo Fisher Scientific) into pEAQ-HT-DEST1 52 and pYeDP60GW 53 for expression in N. benthamiana and S. cerevisiae, respectively. The gene without signal peptide was amplified thanks to IbICS_Rev associated with IbICS_Ecoli_Fw: 5′-ATGTGCTATTCGGCCGTTTTCGGC-3′ from pEAQ-HT-IbICS, ligated into pCR™8/GW/TOPO™ system and cloned by LR recombination into p0GWA 54 for expression in E. coli.…”