Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2-C8) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment. We examined the substrate range of isoprene-degrading bacteria, and also showed that several microbes which cannot use this major plant-produced volatile for growth can nevertheless readily degrade it by co-oxidation. Inhibitor trials identified that octyne and acetylene act as differential inhibitors of isoprene monooxygenase and the monooxygenases of several non-isoprene degraders. Hence, these inhibitors can be used to distinguish isoprene uptake by bone fide isoprene degraders from co-oxidation by, for example, methane utilizers and nitrifiers.image

Automated summary

The study investigated the co-oxidation of various alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium expressing isoprene monooxygenase. The results revealed the broad substrate specificity of this monooxygenase and identified 1-octyne as a potential inhibitor for distinguishing isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria.