Unraveling the differences of the hydrolytic activity of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase: a quantum mechanics-molecular mechanics modeling study

Abstract

Chagas' disease, also known as American trypanosomiasis, is a lethal, chronic disease that currently affects more than 10 million people in Central and South America. The trans-sialidase from Trypanosoma cruzi (T. cruzi, TcTS) is a crucial enzyme for the survival of this parasite: sialic acids from the host are transferred to the cell surface glycoproteins of the trypanosome, thereby evading the host's immune system. On the other hand, the sialidase of T. rangeli (TrSA), which shares 70% sequence identity with TcTS, is a strict hydrolase and shows no trans-sialidase activity. Therefore, TcTS and TrSA represent an excellent framework to understand how different catalytic activities can be achieved with extremely similar structures. By means of combined quantum mechanics-molecular mechanics (QM/MM, SCC-DFTB/Amberff99SB) calculations and umbrella sampling simulations, we investigated the hydrolysis mechanisms of TcTS and TrSA and computed the free energy profiles of these reactions. The results, together with our previous computational investigations, are able to explain the catalytic mechanism of sialidases and describe how subtle differences in the active site make TrSA a strict hydrolase and TcTS a more efficient trans-sialidase.

Scheme 1. Mechanisms Showing the Transfer (Top) And Hydrolysis (Bottom) Reactions

Once the CI_lac is obtained, only TcTS is able to transfer sialic acid to the parasite lactose-containing mucins leading to the MC_lac. If the lactose group in the CI_lac is replaced by a water molecule at the active site of the enzyme, the newly established CI_wat is hydrolyzed by a water molecule to MC_wat. TrSA is a very efficient hydrolase while TcTS preferentially shows trans-sialidase activity.

Figure 1

Representative structure of the TcTS covalent intermediate (CI_wat) used as the starting point for the umbrella sampling simulations. The amino acids included in the classical MM region are shown in green while the QM residues are displayed in light blue sticks. For clarity, only the polar hydrogen atoms are represented.

Scheme 2. Illustration of the Reaction Coordinates Chosen To Simulate the Hydrolysis of the Sialyl-Enzyme Covalent Intermediate

The 2-D reaction coordinate is defined by RC1 and RC2: RC1 = d₃ – d₄ and RC2 = −d₁ – d₂.

Figure 2

Free energy surfaces for the hydrolysis of the CI_wat catalyzed by TcTS (left) and TrSA (right). Results are in kilocalories per mole. The white dashed lines illustrate the minimum energy path to reach MC_wat from CI_wat.

Figure 3

Reaction space for the hydrolysis of the CI_wat during the sialidase activity of TcTS (red) and TrSA (black).

Figure 4

Structures obtained from the QM/MM umbrella sampling simulations corresponding to the covalent intermediate (CI_wat), transition state (TS_wat), and final Michaelis complex (MC_wat), for the hydrolysis of sialic acid catalyzed by TcTS (top) and TrSA (bottom).

Figure 5

Anomeric C–O13 distance (red) and C9 (anomeric C)–C10–O13–C19 dihedral angle (green) along the minimum free energy path in TcTS (top) and TrSA (bottom). Atom names correspond to those in the PDB file.

Figure 6

Free energy profiles for the trans-sialidase (top) and sialidase (bottom) activities catalyzed by TcTS and TrSA.

Figure 7

Relative stabilization pattern of the most relevant active site residues on the TS_wat considering the CI_wat as reference.