The molecular structure of trimethylamine dehydrogenase (TMADH) will be completed at 2.4A resolution and extended to 1.8A resolution. Investigation of the catalytic mechanism will be carried out by difference Fourier studies of crystals modified by substitution and by site-directed mutagenesis. Finally, crystals of the electron transfer flavoprotein (ETF) and its complex with TMADH will be prepared and analyzed. The structure of TMADH from the methylotrophic bacterium W3A1 has been solved at 2.4A resolution and interpreted with an amino acid sequence derived from the electron density map. The protein is a symmetric dimer of Mr 166,000 with each subunit containing a covalently bound FMN, a (4Fe-4S) center and a molecule of ADP. The subunits each contain 3 domains. One domain is a beta 8 alpha 8 parallel beta barrel and contains the FMN and iron-sulfur center. The other two domains contain 5- stranded parallel alpha/beta structures, similar to glutathione reductase, with the ADP moiety lying between them. The DNA sequence of the TMADH gene, which is presently being cloned, will be determined and used to complete the 2.4A structure analysis. The data will then be extended to 1.8A resolution and used for refinement of the structure. Crystals will be studied in various redox states and with substrates and inhibitors bound to them at 2.4A resolution, in order to investigate the mechanism of enzyme action and intramolecular electron transfer. Site-specific mutagenesis of the cloned gene will also be carried out to study the structural and catalytic role of various amino acids. The ETF which serves as the natural electron acceptor for TMADH is a heterodimer of Mr 75,000 containing a single FAD cofactor. Crystals of ETF and its complex with TMADH will be prepared using techniques such as vapor diffusion, microdialysis or free-interface diffusion. The crystals will be analyzed by the multiple isomorphous replacement method and by computer graphics and refinement techniques.
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