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Graphium 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. |
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