Characterizing and Evolving the Propane Monooxygenase for N-Nitrosodimethylamine Biodegradation and Green Chemistry
(in collaboration with William Mohn and Lindsey Eltis of University of British Columbia, Vancouver, and Thomas Wood at Texas A&M)
Recent advancements in analytical detection methods have led to a growing awareness of the presence of N-nitrosodimethylamine (NDMA) in many drinking water sources. NDMA is a member of extremely-potent carcinogens, nitrosamines, whose occurrence in the environment has been linked to decomposition of hydrazine-based rocket fuels and chlorination of water and wastewater.
Bacterial strains expressing specific types of monooxygenases have demonstrated the ability to degrade NDMA, with induction of the propane monooxygenase in Rhodococcus sp. RR1 leading to the fastest rates of degradation. In addition to NDMA, other environmental contaminants such as trichloroethene (TCE), methyl tert-butyl ether (MTBE), and chloroform can be degraded by bacterial strains induced on propane. Despite its apparent versatility as a bacterial enzyme system, very little is known about bacterial propane monooxygenases.
The goal of this project is to characterize and evolve the propane monooxygenase enzyme, to better understand the nature of this enzyme to degrade NDMA and other xenobiotics, and to enhance the transformation rates of those compounds.
Our approach is to first use the fully annotated genome of Rhodococcus sp. RHA1, a strain similar to RR1, to identify and heterologously express the gene cluster coding for propane monooxygenase in a recombinant clones.
The second goal is to purify the components of the propane monooxygenase from the recombinant clones to investigate enzyme activity reconstituted in vitro and for crystallization. Both the recombinant clones and the purified propane monooxygenase will identify the enzymes xenobiotic transformation abilities.
The final goal is to use random and site-specific mutagenesis for directed evolution of the propane monooxygenase in order to improve transformation of xenobiotics. This will be the first instance that protein engineering will be applied to increase the degradation abilities of a propane monooxygenase. Evolved enzymes will be evaluated for green chemistry applications, such as for producing dyes or pharmaceuticals.
Funded by
National Institute of Health.
Transcriptomic evidence for the role of propane monooxygenase in NDMA degradation. Growth of Rhodococcus sp. RHA1 on propane greatly enhances the rate of NDMA degradation. A DNA microarray was used to determine which enzyme is responsible for NDMA degradation under propane growth conditions. The microarray plot (above) shows a 125-fold increase in expression levels of a propane monooxygenase gene (prmA) when RHA1 transcripts from growth on propane are compared to that on pyruvate. These results suggests that a propane monooxygenase is responsible for NDMA degradation. Additional molecular biology strategies, such as qRT-PCR, knockout phenotypes and recombinant clones, will be used to verify these microarray findings.
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