Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase Domain of Human Fatty Acid Synthase

Human fatty acid synthase (hFAS) is a large multienzyme that catalyzes all steps of fatty acid synthesis, which is overexpressed in many cancer cells. Studies have shown that FAS inhibitors exhibit antitumor activity without relevant effects over normal cells. Therefore, the molecular description of active sites in hFAS should stimulate the development of inhibitors as anticancer drug candidates. The malonyl-acetyl transferase (MAT) domain is responsible for loading acetyl-CoA and malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a carrier for fatty acid reaction intermediates. In this work, we have applied computational QM/MM methods at the DLPNO–CCSD­(T)/CBS:AMBER level of theory to study the MAT reaction mechanism. The results indicate that the initial catalytic stage occurs in two sequential steps: (1) nucleophilic attack on the thioester carbonyl group of the substrate through a concerted pathway that involves a Ser-His dyad and (2) tetrahedral intermediate breakdown and release of the free coenzyme A. The Gibbs activation energies for the first and second steps are 13.0 and 6.4 kcal·mol^–1 and 10.9 and 8.0 kcal·mol^–1, whether the substrate transferred to the MAT domain was acetyl-CoA or malonyl-CoA, respectively. Both Met499 and Leu582 form an oxyanion hole that lodges the negative charge of the substrate carbonyl, lowering the first step energetic barriers for both substrates. The mutation of the Arg606 residue by an alanine severely impairs the malonyl transfer reaction, while leading to a kinetic improvement of the transferase activity for acetyl-CoA, which is in agreement with earlier experimental studies. The results from this work encourage future studies that aim for the full comprehension of the MAT catalytic reaction and for the rational design of novel antineoplastic drugs that target this domain

Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase Domain of Human Fatty Acid Synthase

Human fatty acid synthase (hFAS) is a large multienzyme that catalyzes all steps of fatty acid synthesis, which is overexpressed in many cancer cells. Studies have shown that FAS inhibitors exhibit antitumor activity without relevant effects over normal cells. Therefore, the molecular description of active sites in hFAS should stimulate the development of inhibitors as anticancer drug candidates. The malonyl-acetyl transferase (MAT) domain is responsible for loading acetyl-CoA and malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a carrier for fatty acid reaction intermediates. In this work, we have applied computational QM/MM methods at the DLPNO–CCSD­(T)/CBS:AMBER level of theory to study the MAT reaction mechanism. The results indicate that the initial catalytic stage occurs in two sequential steps: (1) nucleophilic attack on the thioester carbonyl group of the substrate through a concerted pathway that involves a Ser-His dyad and (2) tetrahedral intermediate breakdown and release of the free coenzyme A. The Gibbs activation energies for the first and second steps are 13.0 and 6.4 kcal·mol^–1 and 10.9 and 8.0 kcal·mol^–1, whether the substrate transferred to the MAT domain was acetyl-CoA or malonyl-CoA, respectively. Both Met499 and Leu582 form an oxyanion hole that lodges the negative charge of the substrate carbonyl, lowering the first step energetic barriers for both substrates. The mutation of the Arg606 residue by an alanine severely impairs the malonyl transfer reaction, while leading to a kinetic improvement of the transferase activity for acetyl-CoA, which is in agreement with earlier experimental studies. The results from this work encourage future studies that aim for the full comprehension of the MAT catalytic reaction and for the rational design of novel antineoplastic drugs that target this domain

Parameters for Molecular Dynamics Simulations of Manganese-Containing Metalloproteins

A set of geometrical parameters has been determined for single manganese metalloproteins for the AMBER force field, and ultimately to other force fields with a similar philosophy. Twelve (12) models from 9 different single-cluster manganese proteins were optimized and parametrized, using a bonded model approach. Mn-ligand bonds, Mn-ligand angles, and Restrained Electrostatic Potential charges for all the 74 residues in the first metal coordination sphere of each Mn metalloprotein were parametrized. The determined parameters were validated with molecular dynamics simulations and several statistics strategies were used to analyze the results. In addition, to validate the parametrized models, frequency and normal mode calculations were performed and comparisons were obtained for the overall structures both with quantum mechanics and molecular mechanics calculations. Linear and polynomial fittings were performed to estimate Mn-ligand bond force constants for generic manganese centers. Furthermore, averages are proposed for the main Mn-ligand angle interactions of typical manganese coordination centers: axial, square and triangular equatorial planes, and tetrahedral positions, for the different combinations of donor atoms from waters and hard ligands