| Title: | Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45 |
| Author(s): | Sims LP; Lockwood CWJ; Crombie AT; Bradley JM; Le Brun NE; Murrell JC; |
| Address: | "School of Environmental Sciences, University of East Angliagrid.8273.e, Norwich, United Kingdom. School of Chemistry, University of East Angliagrid.8273.e, Norwich, United Kingdom" |
| ISSN/ISBN: | 1098-5336 (Electronic) 0099-2240 (Print) 0099-2240 (Linking) |
| Abstract: | "Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle" |
| Keywords: | Butadienes Carbon/metabolism Hemiterpenes/metabolism Mixed Function Oxygenases/metabolism Oxygenases/metabolism *Rhodococcus/metabolism Actinobacteria isoprene metabolism soluble diiron monooxygenase volatile organic compounds; |
| Notes: | "MedlineSims, Leanne P Lockwood, Colin W J Crombie, Andrew T Bradley, Justin M Le Brun, Nick E Murrell, J Colin eng BB/M011216/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom BB/R002363/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom BB/R013578/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom Research Support, Non-U.S. Gov't 2022/03/15 Appl Environ Microbiol. 2022 Apr 12; 88(7):e0002922. doi: 10.1128/aem.00029-22. Epub 2022 Mar 14" |
