| Recent discoveries in Xanthobacter autotrophicus strain Py2 have allowed the biochemical characterization of a microbial pathway in epoxypropane utilization. Epoxypropane metabolism consists of a nucleophilic attack on the epoxide by CoM, which acts as the metabolic carrier molecule, followed by stereospecific dehydration and carboxylation releasing reduced CoM and acetoacetate. Initial research led to the cloning and heterologous overexpression of the epoxyalkane:CoM transferase, the first enzyme in epoxide metabolism, from X. autotrophicus strain Py2 in Escherichia coli to produce a coenzyme free form of the enzyme. The coenzyme free form of the CoM transferase was then used to produce an enzymatic CoM bioassay to measure the concentration of CoM in any given sample. The enzymatic CoM bioassay was used to show that the inducible propylene and epoxypropane degrading systems are regulated in the same timely manner as the CoM biosynthetic pathway. Further research identified a linear megaplasmid 320 kbp long containing the genes responsible for propylene, epoxypropane, and CoM biosynthesis. To solidify the linear megaplasmid results, X. autotrophicus was subcultured repeatedly in nutrient rich media, and mutants that lacked the ability to grow on propylene as a source of carbon and energy also lacked the linear megaplasmid. Research was then pursued on the heterologously expressed form of the CoM transferase, including elucidation of the pKa of the enzyme bound CoM, identification of the ligands to the zinc center, dissociation constant for CoM with wild type and site directed mutant forms of the enzyme, kinetic parameter determination with CoM and epoxypropane, and identification of the thermodynamic parameters for CoM binding. |
