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Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

ABSTRACT: Trypanosoma cruzi trans-sialidase (TcTS) is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. It catalyzes the transfer of sialic acids from the host's glycoconjugates to the parasite's glycoconjugates. Based on kinetic isotope effect (KIE) studies, a strong nucleophilic participation at the transition state could be determined, and recently, elaborate experiments used 2-deoxy-2,3-difluorosialic acid as substrate and were able to trap a long-lived covalent intermediate (CI) during the catalytic mechanism. In this paper, we compute the KIE and address the entire mechanistic pathway of the CI formation step in TcTS using computational tools. Particularly, the free energy results indicate that in the transition state there is a strong nucleophilic participation of Tyr342, and after this, the system collapsed into a stable CI. We find that there is no carbocation intermediate for this reaction. By means of the energy decomposition method, we identify the residues that have the biggest influence on catalysis. This study facilitates the understanding of the catalytic mechanism of TcTS and can serve as a guide for future inhibitor design studies.

SUBMITTER: Pierdominici-Sottile G

PROVIDER: S-EPMC3779645 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

Pierdominici-Sottile Gustavo G Horenstein Nicole A NA Roitberg Adrian E AE

Biochemistry 20111027 46

Trypanosoma cruzi trans-sialidase (TcTS) is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. It catalyzes the transfer of sialic acids from the host's glycoconjugates to the parasite's glycoconjugates. Based on kinetic isotope effect (KIE) studies, a strong nucleophilic participation at the transition state could be determined, and recently, elaborate experiments used 2-deoxy-2,3-difluorosialic acid as substrate and were able to tra ...[more]

PMID: 22007596

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Project description:Oral transmission of T. cruzi is probably the most frequent mechanism among animals in the wild. In this context, there is a high prevalence of human infections in regions where triatomine infection with T. cruzi is low. This led to the hypothesis that the consumption of raw or undercooked meat from animals infected with T. cruzi could be responsible for the transmission of the infection. Therefore, the general objective of this study was to demonstrate the role of meat consumption from infected animals in the oral transmission of T. cruzi infection. Groups of five female mice Balb/c were fed with muscles obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacity. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies. In all cases, 60–100% of animals were fed meat from mice infected in the acute phase or infected by means of a nasogastric probe with trypomastigotes or amastigotes. These animals developed high parasitemia, and 80% died around day 40 post-infection. The adhesion tests showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS also confirmed that transialidase may be involved in TCT attachment of TCT or invasion of stomach cells. It was concluded that the consumption of meat from infected animals in the acute phase of T. cruzi infection allows transmission of the infection. Similarly, trypomastigotes and amastigotes were able to infect mice when administered orally, while cruzipain was a relevant molecule in this infective process.

Project description:Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.

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Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

Publications

Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

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