The primary purpose of this project is to determine the NMR structure of a component in the toluene-4-monooxygenase complex. Toluene-4-monooxygenase is a three component enzyme system which catalyzes the oxidation of aromatic hydrocarbons, and toluene to p-creosol in particular. The gene sequence for the protein is located in the fourth open reading frame in the operon, so it is therefore it is called toluene-4-monooxygenase, component D (TMOD). The size of the protein is 103 amino acids (11 kDa). At this stage, a set of multinuclear data (15N-HSQC-NOESY, 15N-HSQC-TOCSY, HNCO, HNCA and HN(CO)CA) have been collected and are being analyzed. More triple resonance experiments are due to be collected. These investigations has shown that TMOD yields satisfactory NMR spectra, but has also revealed that the protein may be unstable under certain conditions. A set of homonuclear two-dimensional NMR spectra (COSY, TOCSY, NOESY) has been acquired. Further NMR experiments will be performed using 15N labeled protein.
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