9GN5

EpxF in complex with FAD from Goodfellowiella coeruleoviolacea

Summary for 9GN5

• Entry DOI: 10.2210/pdb9gn5/pdb
• Related: 4N5F
• Descriptor: EpxF, FLAVIN-ADENINE DINUCLEOTIDE, SULFATE ION, ... (5 entities in total)
• Functional Keywords: natural product biosynthesis, flavoenzyme, enzyme mechanism, structure function relationships, oxidoreductase
• Biological source: Goodfellowiella coeruleoviolacea
• Total number of polymer chains: 1
• Total formula weight: 64682.94

Authors

Walter, A.,Kuttenlochner, W.,Eisenreich, W.,Groll, M.,Storch, G. (deposition date: 2024-08-30, release date: 2025-09-10, Last modification date: 2025-10-22)

Primary citation

Walter, A.,Kuttenlochner, W.,Eisenreich, W.,Yao, C.,Groll, M.,Storch, G.
Studies of alpha ', beta '-Epoxyketone Synthesis by Small-Molecule Flavins and Flavoenzymes.
Angew.Chem.Int.Ed.Engl., :e202512568-e202512568, 2025

PubMed Abstract: Epoxomicin is a highly potent natural proteasome inhibitor and the structural scaffold for the anticancer drug carfilzomib. The biosynthesis of its α',β'-epoxyketone warhead involves the flavoenzyme EpxF, but a molecular understanding of the key catalytic reaction cascade remained elusive. Here, we disclose detailed mechanistic insights by characterizing all intermediates in the sequential steps of decarboxylation, desaturation, and epoxidation with synthetic flavins and the flavin-dependent oxidoreductase EpxF. A high-resolution crystal structure of EpxF revealed the architecture of the active site and enabled the identification of key catalytic residues. Exploratory docking based on this structure served as a qualitative tool to guide mutagenesis and rationalize substrate recognition. NMR studies with a C-labeled epoxomicin precursor and structure-based EpxF variants further supported the proposed mechanism. Our integrated approach revealed similarities between synthetic and natural flavin catalysts and offers avenues for developing sustainable biomimetic reactions.

PubMed: 41084917
DOI: 10.1002/anie.202512568

Experimental method

X-RAY DIFFRACTION (2.4 Å)