Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily
| Autor: | Neil L. Wilson, Nga B. Le, Andrew J. Holmes, Mai A Ly, Nicholas V. Coleman, Hitoha E Ogawa, Victoria McCarl |
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| Rok vydání: | 2011 |
| Předmět: |
Mutagenesis (molecular biology technique)
Microbiology Mixed Function Oxygenases Mycobacterium law.invention Ammonia law Alkanes Phylogeny Ecology Evolution Behavior and Systematics chemistry.chemical_classification biology Ammonia monooxygenase Monooxygenase biology.organism_classification Recombinant Proteins Enzyme Biochemistry chemistry Recombinant DNA Original Article Specific activity Heterologous expression Methane Copper |
| Zdroj: | The ISME Journal. 6:171-182 |
| ISSN: | 1751-7370 1751-7362 |
| DOI: | 10.1038/ismej.2011.98 |
| Popis: | The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH(4)) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH(4) and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C(2)-C(4) alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis-this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min(-1) per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C(2)-C(4) alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible. |
| Databáze: | OpenAIRE |
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