# In vivo evidence for glycyl radical insertion into a catalytically inactive variant of pyruvate formate‐lyase

## TL;DR

The study shows that a radical in a key enzyme forms independently of a nearby cysteine pair, challenging previous assumptions about its activation mechanism.

## Contribution

Demonstrates in vivo that glycyl radical formation at G734 is independent of radical transfer to C419, clarifying the enzyme's catalytic mechanism.

## Key findings

- PflBC418A/C419A cannot replace GrcA in restoring formate production in E. coli.
- Glycyl radical formation at G734 is independent of radical transfer to C419 in vivo.
- Radical transfer is intramolecular and the enzyme does not exhibit half-site reactivity.

## Abstract

The dimeric glycyl radical enzyme pyruvate formate‐lyase (PflB; formate acetyltransferase 1) cleaves pyruvate with hypothetical half‐site reactivity to formate and acetyl‐CoA. The radical introduced onto residue G734 of PflB is transiently transferred to C419 of an adjacent cysteine pair (C418/C419) during catalysis, but it is unclear whether glycyl radical formation is dependent on C419 in vivo. We show here that a deficiency in formate production of an Escherichia coli strain synthesizing a PflBG734A variant, but lacking the autonomous glycyl radical cofactor, GrcA, could be restored by reintroducing plasmid‐encoded native PflB, but not by a PflBC418A/C419A variant, indicating that PflBC418A/C419A cannot replace GrcA. Oxygen‐dependent polypeptide cleavage of PflBC418A/C419A indicated stable glycyl radical incorporation; however, these data did not support half‐site reactivity. These in vivo findings demonstrate that glycyl radical formation is independent of subsequent radical transfer from G734 to C419, which occurs intramolecularly.

Impact statementActive, dimeric pyruvate formate‐lyase has a stable radical on a glycine residue, which transiently abstracts a H‐atom from a cysteine, generating a catalytic thiyl radical. Glycyl radical generation is independent of glycine‐to‐cysteine radical‐transfer in vivo. Radical‐transfer is intramolecular and the enzyme does not appear to exhibit half‐site reactivity.

Active, dimeric pyruvate formate‐lyase has a stable radical on a glycine residue, which transiently abstracts a H‐atom from a cysteine, generating a catalytic thiyl radical. Glycyl radical generation is independent of glycine‐to‐cysteine radical‐transfer in vivo. Radical‐transfer is intramolecular and the enzyme does not appear to exhibit half‐site reactivity.

Dimeric pyruvate formate‐lyase cleaves pyruvate using a radical‐based mechanism. G734 serves as a radical storage location, and the radical is transferred to the catalytic C419 residue. Mutation of the C418‐C419 pair causes loss of enzyme activity, but does not impede radical introduction onto G734. Therefore, cis‐ but not trans‐radical transfer occurs in vivo and radical storage is independent of catalysis.