# Structural and Functional Validation of Pseudomonas Savastanoi Ethylene Forming Enzymes Reveals Flexibility in 2‐Oxoglutarate Binding Mode and Conformation

## TL;DR

Researchers studied a bacterial enzyme that produces ethylene and found that small changes in the enzyme's structure can affect how it binds to a key molecule, which might influence its function.

## Contribution

The study reveals structural and functional flexibility in 2OG binding modes of PsEFE variants from Pseudomonas savastanoi.

## Key findings

- NMR assays confirm that PsEFE variants from P. savastanoi produce ethylene from 2OG.
- Crystallographic analysis shows similar overall structure but an atypical 2OG binding mode in one PsEFE variant.
- Variants exhibit different product ratios and potential metal ion sensing capabilities.

## Abstract

Pseudomonas savastanoi pv. phaseolicola PK2 employs an Fe(II)‐dependent ethylene/succinate‐forming enzyme (PK2 PsEFE) to produce ethylene from 2‐oxoglutarate (2OG). Here we report NMR‐based assays showing that the putative P. savastanoi pv. glycinea PsEFE, which differs from PK2 PsEFE by a single residue, and the P. savastanoi pv.1449B PsEFE, which differs from PK2 PsEFE by 28 residues and a C‐terminal 13‐residue truncation, catalyze ethylene production from 2OG. Like the PK2 PsEFE, they catalyze oxidation of naturally occurring 2OG derivatives to give alcohol and diacid products. Crystallographic analysis demonstrates that the overall fold and active site of 1449B PsEFE is similar to that of PK2 PsEFE. Interestingly, 2OG was observed to adopt an atypical inverse metal ion binding mode in complex with 1449B PsEFE:Mn in which its 2‐oxoacid group is positioned to interact with the guanidinium group of R277, but not the Mn ion, which substitutes for catalytically active Fe(II). Together with reported crystallographic results, this observation indicates that 2OG metal ion binding modes and conformations at the active sites of 2OG oxygenases can vary, possibly in a functionally or disease relevant manner.

Studies with naturally occuring variants of the Pseudomonas savastanoi ethylene‐forming enzyme (PsEFE) inform on the effect of residues on turnover and product ratio. Crystallographic studies imply a potential for metal ion sensing.© 2026 WILEY‐VCH GmbH