Substrate‐dependent dynamics of UDP‐galactopyranose mutase: Implications for drug design

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, a precursor of the cell surface b-galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site-directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.

Structure, mechanism, and dynamics of UDP-galactopyranose mutase

“…MD simulations have also been used to study substrate-associated active site motion in TcUGM [58]. Classical and accelerated MD simulations agree with the proposed hypothesis that ligand binding influences the open-closed equilibrium of the mobile flaps.…”

“…Classical and accelerated MD simulations agree with the proposed hypothesis that ligand binding influences the open-closed equilibrium of the mobile flaps. After extensive MD simulations of both substrate-free and substrate-bound TcUGM, it was observed that both flaps open in the absence of ligand, thus creating a channel for ligand uptake [58]. When UDP-Gal p is bound, intramolecular interactions help maintain both flaps closed.…”

Trypsinogen activation as observed in accelerated molecular dynamics simulations

“…The aMD approach is especially attractive because it does not require a priori knowledge of the reaction path. This technique has been successfully used during the last years by our group to sample conformational changes occurring on the micro‐ to millisecond time scales …”

“…This technique has been successfully used during the last years by our group to sample conformational changes occurring on the micro-to millisecond time scales. [28][29][30][31] The implementation of aMD allows modification of both the total potential energy (V total ) of the system as well as the dihedral potential energy (V dihed ). It is possible to modify only the first one, only the second one or both of them at the same time; that is, the dihedral energy is doubly modified or more accelerated than other terms.…”

Multiple functionalities of reduced flavin in the non-redox reaction catalyzed by UDP-galactopyranose mutase

“…The presence of a covalent flavin-N5-adduct was demonstrated in eUGMs [41] and the structures of the free enzyme and in complex with UDP-Gal p in the oxidized and reduced state have been elucidated [42–45]. These structures revealed conformation changes in the active site flaps, which open in the absence and close in the presence of substrate.…”