Structure of TMADH

Early crystallographic studies of TMADH provided data from two derivatives at 6 resolution that revealed the domain structure and certain elements of secondary structure (Lim et al., 1982 Lim et al., 1984). Higher resolution data at 2.4 resolution have been collected and the structure solved by the multiple isomorphous replacement method with anomolous scattering (Lim et al., 1986). Analysis of the diffraction pattern lead to the identification of ADP as the third cofactor in TMADH. At the time the 2.4 data set was analysed, there was no sequence information available for TMADH (Lim et al., 1986), except for a 12 residue peptide which contained the covalently bound flavin (Kenney et al., 1978). Gas-phase sequencing of isolated peptides initially provided 80% of the primary sequence of 

The chemically determined sequence was found to be approximately 80% identical with the ix-ray deduced sequence (Barber et al., 1992). Aeeounts of some of the earlier crystallographic studies have been reviewed (Steenkamp and Mathews, 1992). The structure of TMADH has now been solved at 2.4 resolution (Brookhaven code 2TMD) and more reeently at 1.7 resolution (Mathews et al., unpublished) and these studies were aided by the full determination of the primary sequence through gene sequeneing methods (Boyd et al., 1992). 

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FIGURE 3. Ca trace illustrating the structural similarity between the small domain of TMADH (solid line) and the NADPH-binding domain of glutathione reductase (dashed line). 

A major handicap to our detailed rmderstanding of the electron transfer reactions between TMADH and ETF is the lack of a crystallographic structure for ETF. Crystals of ETF have been isolated (White et al., 1994), but to date no structure for the protein has been reported. A homology model for ETF, however, has been constructed based on the crystallographic structure of human ETF (Roberts et al., 1996), and this model has been used to create a model of the electron transfer complex formed between TMADH and ETF (Chohan et al., 1998) (Figure 7). ETF comprises two subunits, which in turn form three domains. Domain I comprises the N-terminal region of the a-subunit, domain II comprises the C-terminal... 

The foeus of this chapter is the soluble electron transfer complex formed between the nieotinamide-independent trimethylamine dehydrogenase (TMADH) and eleetron transferring flavoprotein (ETF). Recent studies of this physiological electron transfer complex have provided invaluable insight into (i) the mechanisms of inter and intraprotein electron transfer between flavin and Fe/S centers, (ii) the role of dynamics in interprotein electron transfer and (hi) quantum meehanieal mechanisms for the cleavage of substrate C-H bonds and the subsequent transfer of reducing equivalents to flavin redox centers. Brief mention is made of early structural and cofactor analyses for this redox system, but more detailed accounts of this work can be found in earlier reviews on the subjeet (e.g. Steenkamp and Mathews, 1992). 

FIGURE 7. Proposed large-scale structural reorganisation of ETF on binding to TMADH. Schematic representations of A, the ieT-inactivei complex, and B, the ieT-activei complex. The corresponding structures (Ca trace for protein) are shown in C and D, respectively. 

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