The molecular structure of Trimethylamine Dehydrogenase (TMADH) from the methylotrophic bacterium W3A1 will be studied by X-ray diffraction. TMADH is a complex iron-sulfur flavoprotein of 147,000 daltons composed of two non-identical subunits of about 75,000 daltons each. It contains one Fe4S4 cluster and one covalently bound FMN coenzyme per molecule. The enzyme catalyzes the oxidation of trimethylamine to dimethylamine and formaldehyde with subsequent electron transfer to the artificial acceptor phenazine methosulfate. When reduced by substrate the enzyme exhibits very strong spin coupling between the reduced cofactors accompanied by an apparent conformation change. Crystals of TMADH are monoclinic, space group P21 with cell dimensions a=147.63A, b=71.96A, c=83.66A and Beta=97.64A. The crystals diffract to 2.0A resolution. The structure will be analyzed by the isomorphous replacement method first at 6.0A and then at 2.4A resolutions. The 6.0A map will be studied to identify the domain and subunit structure of the enzyme. Native anomalous scattering data will be analyzed to locate the iron-sulful cluster. The 2.4A map will be used to trace the polypeptide chain and locate the orientation of the side chains and of the cofactors. A model of the protein will be constructed on the MMS-X molecular graphics system. In order to study the enzymatic mechanism, various substrates, inhibitors and allosteric effectors will be bound to the enzyme and analyzed by difference Fourier methods. Finally, the structure will be refined to 2.0A resolution by the Hendrickson-Konnert restrained least squares procedure.
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