br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Knowledge of the molecular basis of an inherited genetic disease is crucial for understanding the disease pathology and, ultimately, for therapeutic design. Several recent reports have stressed the need for physical characterization of disease-associated protein variants [10], [18], emphasizing the inability of structural data alone to reveal the functional impact of a particular mutation. Previous studies on the effect of PDE-related mutations on ALDH7A1 activity used either transient UM171 [3] or analysis of crude lysate from heterologous expression [19], [20]. In this study, we have conducted detailed biochemical and biophysical analyses on the purified variant proteins, in an effort to understand how these previously uncharacterized mutations, positioned at or near the interfaces of the ALDH7A1 quaternary structure, impact activity and structure. Whereas previous structural studies suggested a tetrameric quaternary structure for ALDH7A1 [7], [8], [9], our results indicate more complex self-association behavior. Analysis of sedimentation-equilibrium data (Table 1) suggests that ALDH7A1 will be predominately dimeric at standard enzyme-assay concentrations (i.e., ≈1 μM), with only minor amounts of tetramer present. In this respect, ALDH7A1 resembles ALDH3A1 [21] and ALDH4A1 [22], previously shown to be dimers. Whether our in vitro sedimentation measurements accurately describe the self-assocation behavior of ALDH7A1 in vivo is unknown. For example, it is possible that molecular crowding effects could increase the affinity of dimers to form tetramers in vivo. Nevertheless, the ALDH7A1 activity in vitro scales with enzyme concentration between 0.5 and 4.0 μM, a range over which the percentage of tetramer increases from 3% to 17% of the total protein. This finding suggests that the specific activities of the dimeric and tetrameric forms may be comparable. Another possibility is the dimers have low or no activity, and the solution conditions of the activity assay promote formation of highly active tetramers. In this mechanism, the binding of substrates to the enzyme may facilitate tetramerization. Additional studies are needed to distinguish between these scenarios. All examined PDE-related mutations, proximal to the protein-protein interfaces of ALDH7A1, abolish enzyme activity. Neither P78L nor G83E is expressed in appreciable amounts as soluble protein in E. coli, therefore, they were not included in these studies. Although the remaining variants (A129P, G137V, G138V, A149E, G255D, G236E) express well and are soluble, they exhibit altered quaternary structure. Whereas the wild-type protein is primarily dimeric at low μM concentrations, the variants reside in monomer-dimer equilibria, characterized by dissociation constants between 0.7 and 5 μM. The total inability of the variants to catalyze the oxidation of AASAL implies that the monomers are inactive and, moreover, that their self-association does not restore activity. The latter conclusion suggests that the association surfaces of the mutated enzymes are sufficiently altered so as to render formation of the wild-type dimer-interface unfavorable. Parenthetically, our results are reminiscent of a previous ALDH1 study in which simultaneous mutation of two interface residues disrupted tetramer formation and severely compromised enzymatic activity [23]. This behavior is evidently not universal, given that certain mutations that disrupt the hexameric form of bacterial ALDH4A1 do not affect enzymatic activity [24].
Conclusions
Conflict of interest
Acknowledgements Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM093123.