Graduation date: 2007Graphium sp., a eukaryotic alkanotroph, is able to oxidize small-molecular weight gaseous n-alkanes, diethyl ether and the branched ether, methyl tert butyl ether (MTBE). However, information regarding the biochemistry of fungal-mediated alkane and ether metabolism is limited, and questions regarding the identity of alkane oxidation catalysts and the genetic underpinnings of alkane metabolism are unresolved. The objectives of this investigation were to refine the pathway and the regulation of MTBE metabolism, to further define the substrate range and to identify and characterize the hydroxylase responsible for alkane and ether oxidation in this Graphium species. We found that Graphium oxidizes MTBE through a novel variation of an existing pathway first described in n-alkane-grown Mycobacterium vaccae JOB5. However, the fungus is unable to utilize the products of MTBE metabolism, resulting in the accumulation of potentially toxic intermediates. We also found that the regulatory effects of MTBE oxidation intermediates proposed for other MTBE- degrading organisms do not impact Graphium-mediated MTBE metabolism and thus are not universally relevant mechanisms for MTBE-degrading organisms. Given that Graphium is able to degrade MTBE and diethyl ether, we investigated the ability of the fungus to degrade environmentally relevant cyclic ethers including tetrahydrofuran (THF) and 1,4-dioxane (14D). Our investigation of cyclic ether metabolism revealed that Graphium sp. utilizes THF as a sole source of carbon and energy under aerobic conditions via the THF metabolic pathway used by Rhodococcus ruber and two Pseudonocardia strains. Although Graphium sp. was unable to grow on 14D, it was able to cometabolize this compound after growth on either THF or alkanes. The results of our investigations regarding cyclic ether and MTBE metabolism suggested that the metabolic pathways that process these compounds are superimposed on the alkane oxidation pathway. Because monooxygenase-catalyzed substrate activation is both the first and the rate- determining step of these pathways, an additional aim of this investigation was to identify, clone and characterize the gene encoding the alkane monooxygenase from this Graphium sp. Prokaryotic alkanotrophs oxidize alkanes mainly through diiron and copper-containing monooxygenases. Unlike prokaryotes, in Graphium sp., we found that the initial oxidation of alkanes is catalyzed by a cytochrome P450 alkane monooxygenase. This is the first report of a cytochrome P450 monooxygenase that is able to oxidize gaseous n-alkanes. To further characterize this novel enzyme, we also estimated the regiospecificity of alkane oxidation and determined that although the majority of hydroxylation events result in terminal carbon oxidation, a significant portion of these events result in subterminal oxidation. Subterminal oxidation can produce metabolites that are unpalatable and possibly toxic. Taken as a whole, the results of these investigations significantly extend the known growth substrates and lend insight into the biochemical foundations and genetic underpinnings that facilitate gaseous n-alkane and ether oxidation by this versatile fungus.