Structural and evolutionary insights into the isoprene monooxygenases

Isoprene, a highly reactive biogenic volatile organic compound (VOC) emitted by terrestrial 34 vegetation, influences atmospheric chemistry but its microbial degradation remains poorly understood. 35 Aerobic degradation begins with isoprene monooxygenase (IsoMO), a multicomponent di-iron 36 monooxygenase encoded by the isoABCDEF cluster, with isoGHIJ supporting downstream steps. We 37 analysed iso gene clusters from eleven confirmed isoprene degraders, reconstructed gene-by-gene 38 phylogenies, and generated structural models of IsoMO components using mainly AlphaFold2. IsoA, 39 IsoB, and IsoE formed a highly conserved α₂β₂γ₂ monooxygenase core (IsoMO core) whose predicted 40 architecture and closely resembled the soluble methane monooxygenase (sMMO) hydroxylase, 41 revealing a shared di-iron catalytic framework adapted to distinct hydrocarbon substrates. IsoA was the 42 most conserved subunit and remains a reliable molecular marker for isoprene degradation. This work 43 presents the first detailed structural model of an IsoMO core and reveals its deep relationship to other 44 soluble di-iron monooxygenases. Together these results provide a molecular foundation for future 45 mechanistic, ecological and inhibitor based studies linking enzyme-level specificity to microbial 46 control of isoprene turnover under changing climate conditions.