The ethylene-forming enzyme (EFE) has long been considered as a constitutively active enzyme of ethylene biosynthesis in plants. However, emerging evidence suggests that ethylene production is dynamically regulated under sudden environmental changes, such as abiotic stresses. We hypothesize that EFE serves as a gatekeeper with a finely tuned regulatory mechanism responsive to varying plant growth conditions.
In this study, we explored the protein-level regulation of EFE using a combination of biochemical and plant-based approaches. Our findings revealed that EFE undergoes redox-controlled disulfide dimerization, modulating its function and activity both in vitro and in planta. Interestingly, we identified that EFE is also subject to glutathionylation and cysteinylglycine modification at the same cysteine residue responsible for dimerization. Unlike dimerization, these modifications do not impact enzyme activity.
To elucidate the molecular basis of these regulatory mechanisms, we employed X-ray crystallography, spectroscopy, site-directed mutagenesis, and molecular dynamics simulations. Our structural analyses revealed that EFE’s substrate-binding pocket resembles that of 2-oxoglutarate (2OG)-dependent oxygenases, though EFE does not utilize 2OG as a co-substrate. The conserved 2OG-binding residues in EFE play a critical role in substrate binding and catalysis by driving a conformational transition from an open to a closed state. Notably, dimerization stabilizes the enzyme in its open conformation, restricting this conformational shift, whereas cysteine modifications inhibit dimerization without affecting catalytic efficiency.
This is the first report of protein-level regulation of EFE, providing new insights into the fine-tuning of ethylene biosynthesis. As ethylene is a pivotal hormone governing various aspects of plant growth and development, our findings could pave the way for advanced agricultural applications, including stress resilience and postharvest management.