NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Abstract

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.

Evidence of Ternary Complex Formation in Trypanosoma cruzi trans-Sialidase Catalysis

“…The second cleft of TS was suggested previously (25, 44) because binding of the acceptor substrate was only achieved when the donor substrate was present (23,24). Such an argument is strengthened by the observation that the inactive mutant TS D247A did not display the second cleft during the MD simulations, which suggests that binding of the acceptor substrate is imperative for the trans-sialidase reaction to take place.…”

“…Moreover, experimental evidence for such plasticity arose from the observation that the non-functional variant of the trans-sialidase (TS Y342H ) (22) undergoes conformational changes upon sialoside binding, suggesting the opening of a second binding site that accommodates a ␤-Galp moiety (6). Active site rearrangement following the binding of sialoside was further proposed for the active TcTS (23). Results of TS Y342H incubated with ␣2,6-sialyllactose in the presence of lacto-N-tetraose show that incorrect fitting of sialoside into the binding site of TS Y342H does not trigger ␤-Galp binding, which corroborates this hypothesis (6).…”

Assembly of Anionic Conjugated Polymer with 6‐O‐Modified PNP‐β‐Galactoside for Fluorescence Logic‐signal‐based Multiplex Detections of Enzymes

“…[9] The typical methods for single type enzyme assay include pH stat, [10] electrochemical techniques, [11] NMR, [12] calorimetry, [13] UV-vis and fluorimetry; [14] however, there is still a need to develop a more sensitive and universal method for multiplex detections of enzymes in one tube. Our previous work shows that CPs can be used to realize multiplex detections of nucleases.…”

A 1H STD NMR spectroscopic investigation of sialylnucleoside mimetics as probes of CMP-Kdn synthetase

“…We have had a longstanding interest [1][2][3] in the sialic acid family of carbohydrates, as well as sialic acid recognising proteins (SARPS), particularly enzymes, that are involved in their biosynthesis and degradation, and NMR spectroscopic methods for the investigation of enzymecatalysed reactions [4,5]. It is well known that nucleotide synthetases are essential for the biosynthesis of activated carbohydrates, commonly referred to as glycosyl donors.…”

“…These Sch. 1 The biosynthesis of CMP-sialic acids catalysed by CMP-Kdn synthetase studies have shed some light on key interactions between various nucleotides and the enzyme. NMR spectroscopy has become a valuable tool in the investigation of the elucidation of ligand-biomolecule interactions [22,23].…”