RECONCILING MOLECULAR DYNAMICS RESULTS WITH EXPERIMENTAL EVIDENCE. THE CASE OF MMACHC, A VITAMIN B12 ENZYME

MMACHC is an enzyme that is crucially involved in the metabolic processing of the vitamin B12 (cobalamin, cbl). The enzyme binds the vitamin and, with the help of co-ligands, it removes the variable upper ligand of cbl, to prepare it for its physiological role. Many MMACHC pathological mutations are known to cause severe symptoms (cblC disease). We have recently studied 1 the effect of a point mutation, R161Q, that is predicted to destabilize the protein. We used several biophysical techniques to compare the structural stability, cbl binding and the enzymatic activity of the recombinant wt and R161Q forms. In parallel to the experimental activity, we modeled the MMACHC and the R161Q mutant in complex with methyl-cbl and with glutathione, an important co-ligand. We performed equilibrium MD simulations as well as free energy approaches, to compare the affinities of the mutant to that of the wt MMACHC. A critical assessment of different binding free energy techniques was an interesting side result. The study of the respective apo forms revealed unexpected results that suggest a high structural sensitivity to solvent conditions. In fact, we observed a very dynamic wt MMACHC, with a high RMSD with respect to the crystallographic structure. Adding malonate ions, present in the crystallographic structure, resulted in a more stable wt and in a more destabilized mutant. Overall, the application of modelling methods to the MMACHC, a protein that features large, flexible and functional loops and is heavily ligand-stabilized, is producing interesting insights but has also revealed significant challenges.