TROF-EM · Tailoring Rieske Oxygenase Function through Electron Transfer Mapping and ML-Guided Design for Enhanced Biocatalysis

Enzyme catalysis is a key technology at the heart of industrial biotechnology, expected to drive economic growth and industrial leadership in the EU, while addressing social, environmental, and economic needs. Despite its growing impact, the full potential of this technology has yet to be realised. TROF-EM aims to bridge the gap between the potential of enzyme catalysis and its application in the sustainable synthesis of pharmaceuticals for Rieske oxygenases (ROs). These enzymes perform highly selective C–H functionalisations through a multi-step electron transfer (ET) cascade, ultimately enabling the formation of reactive intermediates that drive diverse oxidative transformations. Despite their promise, industrial use is limited by poor catalytic efficiency and incomplete understanding of ET mechanisms and protein–protein interactions (PPIs). TROF-EM will address these challenges by integrating enzyme discovery, structural biology, computational modelling, machine learning (ML), protein engineering, and biochemical analysis to decipher how ET and PPIs govern RO activity and selectivity, thereby producing a first-in-class RO biocatalyst tailored for industrial application. To achieve this, TROF-EM pursues three objectives: (i) investigate how molecular recognition, active-site architecture, and PPIs affect ET efficiency and catalysis; (ii) apply ML-guided mutagenesis to generate RO variants with improved efficiency and reduced uncoupling; and (iii) develop a self-sufficient fused RO with enhanced catalytic performance. TROF-EM will foster interdisciplinary collaboration, supported by leading experts in biocatalysis, structural biology, and computational chemistry, to generate insights into ET in multi-component enzymes and deliver novel biocatalysts for efficient late-stage functionalisation of complex drug scaffolds, offering greener, cost-effective alternatives to conventional chemical synthesis and accelerating drug discovery.