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.