Purification and characterization of the isoprene monooxygenase from Rhodococcus sp. strain AD45

Sims, Leanne P., Lockwood, Colin W. J., Crombie, Andrew T., Bradley, Justin M., Le Brun, Nick E. and Murrell, J. Colin (2022) Purification and characterization of the isoprene monooxygenase from Rhodococcus sp. strain AD45. Applied and Environmental Microbiology, 88 (7). ISSN 0099-2240

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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.

Item Type: Article
Additional Information: Funding: The work on this project was funded through an ERC Advanced Grant to J.C.M. (694578; IsoMet), the UKRI Biotechnology and Biological Sciences Research Council (BB/M011216/1 and BB/R002363/1 to N.E.L.B., BB/R013578/1 to N.E.L.B.), and the Leverhulme Trust (Early Career Fellowship ECF-2016-626 to A.T.C.).
Uncontrolled Keywords: actinobacteria,isoprene metabolism,soluble diiron monooxygenase,volatile organic compounds,applied microbiology and biotechnology,food science,biotechnology,ecology,sdg 13 - climate action ,/dk/atira/pure/subjectarea/asjc/2400/2402
Faculty \ School: Faculty of Science > School of Chemistry
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Depositing User: LivePure Connector
Date Deposited: 23 Mar 2022 13:30
Last Modified: 06 May 2022 03:54
URI: https://ueaeprints.uea.ac.uk/id/eprint/84237
DOI: 10.1128/aem.00029-22

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