# Tandem ketone reduction in pepstatin biosynthesis reveals an F420H2–dependent statine pathway

## TL;DR

Scientists discovered a new pathway for making pepstatins, using a special enzyme to create key statine parts.

## Contribution

The study reveals a novel F420H2-dependent oxidoreductase pathway for statine biosynthesis in pepstatin production.

## Key findings

- PepI catalyzes the tandem reduction of β-keto intermediates to form two statine residues in pepstatin.
- The biosynthesis involves an unconventional gene cluster with nonribosomal and polyketide synthase genes.
- Structural and mutagenesis studies identify critical residues in F420H2-dependent oxidoreductase function.

## Abstract

Pepstatins are potent inhibitors of aspartic proteases, featuring two statine residues crucial for target binding. However, the biosynthesis of pepstatins, especially their statine substructure, remains elusive. Here, we discover and characterize an unconventional gene cluster responsible for pepstatin biosynthesis, comprising discrete nonribosomal peptide synthetase and polyketide synthase genes, highlighting its trans-acting and iterative nature. Central to this pathway is PepI, an F420H2-dependent oxidoreductase. The biochemical characterization of PepI reveals its role in the tandem reduction of β-keto pepstatin intermediates. PepI first catalyzes the formation of the central statine, then produces the C-terminal statine moiety. The post-assembly-line formation of statine by PepI contrasts with the previously hypothesized biosynthesis involving polyketide synthase ketoreductase domains. Structural studies, site-directed mutagenesis, and deuterium-labeled enzyme assays probe the mechanism of F420H2-dependent oxidoreductases and identify critical residues. Our findings uncover a unique statine biosynthetic pathway employing the only known iterative F420H2-dependent oxidoreductase to date.

Pepstatins are potent inhibitors of aspartic proteases, featuring two statine residues crucial for target binding, however, their biosynthesis is elusive. Here, the authors discover and characterize an unconventional gene cluster responsible for pepstatin biosynthesis, and characterize the role of PepI, an F420H2-dependent oxidoreductase catalysing the tandem reduction of β-keto pepstatin intermediates.