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

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.