# A Bifunctional Cytochrome P450 Enzyme Catalyzes Hydroxylation and Aryl‐Aryl Ether Formation in the Biosynthesis of Emestrin

## TL;DR

Scientists discovered a new enzyme that can perform two chemical reactions during the production of a complex natural compound called emestrin.

## Contribution

The discovery of a bifunctional P450 enzyme (EmeO) that forms aryl-aryl ether bonds and hydroxylates aromatic rings during emestrin biosynthesis.

## Key findings

- EmeE and EmeR add hydroxyl groups at specific positions on the diketopiperazine ring.
- EmeO is a bifunctional enzyme that forms a lactone ring via aryl-aryl ether bond and hydroxylation.
- This is the first report of such enzymatic reactions in emestrin biosynthesis.

## Abstract

Emestrins, a subgroup of epipolythiodioxopiperazines, are originated from cyclo‐l‐Phe‐l‐Phe and feature a dihydrooxepine ring. They contain typically a 15‐membered lactone ring with an aryl‐aryl ether linkage. Despite considerable progress in elucidating epipolythiodioxopiperazine biosynthesis, the enzymatic mechanism for the ether bond formation in emestrins remains uncharacterized. We identified a putative gene cluster (eme) in the fungus Emericella quadrilineata with three unknown P450 enzymes, EmeE, EmeR, and EmeO. Gene deletion, feeding experiments, and in vitro assays proved that EmeE and EmeR install regioselective and stereospecific hydroxyl groups at the ß‐positions of the diketopiperazine ring. EmeO acts as a bifunctional enzyme for the construction of the lactone ring via an aryl‐aryl ether bond formation and simultaneous hydroxylation between phenolic and nonphenolic aromatic rings. To the best of our knowledge, such enzymatic reactions have not been reported prior to this study.

Conversion of emestrin J (5) to emestrin (1) by three P450 enzymes from the cluster (eme) in Emericella quadrilineata. EmeO acts as a bifunctional enzyme for the construction of the 15‐membered lactone ring via an aryl‐aryl ether bond formation and simultaneous hydroxylation between phenolic and nonphenolic aromatic rings, while EmeE and EmeR install regioselective and stereospecific hydroxyl groups at the ß‐positions of the diketopiperazine ring.