Chagas'disease (American Trypanosomiasis) affects 16-18 million people in Latin America. It has a 25-30% death rate among infected people and causes between 45,000 and 50,000 deaths a year. The flagellate protozoan parasite Trypanosoma cruzi, the causative agent of Chagas'disease is transmitted to humans by bites of blood-feeding """"""""Assassin bugs"""""""". The most common agent is Triatoma infestans (vinchucas in Argentina, or barbeiros in Brazil). The enzyme trans-sialidase in T.cruzi catalyzes the transfer of sialic acids from host aloglycoconjugates to other parasite glycoconjugates and has a critical role in virulence of T.cruzi, the etiological agent of Chagas'disease. T.cruzi trans-sialidase (TcTS) is a validated target for inhibition with therapeutic possibilities for the cure for this lethal chronic disease. TcTS is also of interest due to its strong similarity in sequence and structure to a strict hydrolase, Trypanosoma rangeli sialidase (TrSA). An extensive comparison of the two will reveal structural requirements for sialyl-transferase activity and also be a guide for future efforts to transform sialidases into trans-sialidases that are of synthetic value. This theoretical study will focus on the reaction mechanisms for hydrolysis reactions of TcTS and TrSA and sialyl-transfer reaction of TcTS using hybrid (mixed quantum and classical, QM/MM) methods on entire enzyme structures. There is a methods development part of this proposal associated with two different QM/MM implementations, one native to the program Amber, and one that links Amber with the QM program Gaussian, via a program we designed name Pupil. Molecular dynamics (MD) simulations on various X-ray crystal structures of the enzymes which correspond to different points on the reaction paths will also be performed. Critical active site interactions and dynamical properties for sialyl-transfer reaction will be uncovered analyzing MD simulations of wild type and mutant enzymes and comparing the results. We will also perform extensive research into inhibitor design for TcTS, using a combination of docking, free energy calculations and mechanism-based leads. Public Health Relevance: A broader impact of the present research is the contribution to the understanding of the general principles of catalysis. From a synthetic point of view, trans-sialidases can be used to perform complex syntheses of O-linked and S-linked glycoconjugates of industrial and medicinal value. A deeper understanding of the mechanisms of the enzymes could be a guide for inhibitor design studies of the entire family.
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