Decoding the Cytochrome P450 Catalytic Activity in Divergence of Benzophenone and Xanthone Biosynthetic Pathways

Abstract

Plant benzophenones and xanthones have an impressive spectrum of biological activities. Here, we characterized three cytochrome P450 (CYP) enzymes from the Garcinia xanthochymus tree that convey 2,4,6-trihydroxybenzophenone (2,4,6-triHB) toward the diversification between the biosynthesis of benzophenones and xanthones. The CYP3 catalyzes the transformation of 2,4,6-triHB to 2,3′,4,4′,6-pentahydroxybenzophenone (2,3′,4,4′,6-pentaHB) through two consecutive hydroxylations, while the paralog CYP1 catalyzes the synthesis of 1,3,7-trihydroxyxanthone (1,3,7-triHX). To understand the product diversity, we performed homology modeling and active site-directed reciprocal mutations on CYP3. The mutant enzyme assays revealed the key role of the V375 residue. Although the V375A mutation favors the 1,3,7-triHX formation, the V375L enhances further the yield of 2,3′,4,4′,6-pentaHB. The engineered triple mutant CYP3-V375L/S479A/K480H demonstrated an improved product specificity and catalytic transformation toward 2,3′,4,4′,6-pentaHB. Our results represent an advance in the metabolism of benzophenones and xanthones, laying the foundation for discovery of downstream genes and even the production of new derivatives. The engineering of CYP enzymes that stand in divergence of biosynthetic pathways for benzophenones and xanthones provides the basis for designing new drugs and facilitates future synthetic biology applications.

Single-cell RNA-seq based elucidation of the antidepressant hyperforin biosynthesisde novoin St. John’s wort

“…By mining the H. perforatum genome, we identified a cluster comprising 11 annotated aromatic-prenyltransferase genes bearing high homology to known enzymes acting in biosynthesis of prenylated xanthones 39 . A blueprint of prenylated-xanthone metabolism in Hypericum species [40][41][42] indicates analogies with the proposed model of hyperforin biosynthesis involving the recruitment of dedicated CoA ligases, type-III polyketide synthases and aromatic prenyltransferases that differ in the starting polyketide unit and the role of oxidative cyclizations in the xanthone pathway 43,44 (Figure S6). Toward determining their functionality and potential contribution to hyperforin biosynthesis in the absence of available functionalcharacterization approaches in H. perforatum, the 11 predicted prenyltransferase genes were individually cloned and co-expressed in yeast together with the known CoA ligase (HpCCL2) and polyketide synthase (HpPKS2) from HpBGC1 20 .…”