Structural and evolutionary insights into the isoprene monooxygenases.

Isoprene, a highly reactive biogenic volatile organic compound emitted by terrestrial vegetation, influences atmospheric chemistry but its microbial degradation remains poorly understood. Aerobic degradation begins with isoprene monooxygenase (IsoMO), a multicomponent di-iron monooxygenase encoded by the isoABCDEF cluster, with isoGHIJ supporting downstream steps. We analysed iso gene clusters from 11 confirmed isoprene degraders, reconstructed amino acid sequence phylogenies, and generated structural models of IsoMO components using mainly AlphaFold2. IsoA, IsoE, and IsoB formed a highly conserved α₂β₂γ₂ monooxygenase core (IsoMO core) whose predicted architecture and closely resembled the soluble methane monooxygenase hydroxylase, revealing a shared di-iron catalytic framework adapted to distinct hydrocarbon substrates. IsoA was the most conserved subunit and remains a reliable molecular marker for isoprene degradation. This work presents the first detailed structural model of an IsoMO core and reveals its deep relationship to other soluble di-iron monooxygenases. Together these results provide a molecular foundation for future mechanistic, ecological and inhibitor-based studies linking enzyme-level specificity to microbial control of isoprene turnover under changing climate conditions.