Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration

Abstract

The trans-sialidase from Trypanosoma cruzi is a member of the sialidase superfamily that functions as a sialidase in the absence of a carbohydrate acceptor. We have used 1H nuclear magnetic resonance (NMR) spectroscopy to investigate the stereospecificity of the hydrolysis of two substrates, namely, 4-methyl-umbelliferyl-N-acetylneuraminic acid and α(2-3)-sialyllactose, catalyzed by a recombinant T. cruzi trans-sialidase. We demonstrate that, in aqueous solution, the thermodynamically less stable α-form of N-acetylneuraminic acid is the initial product of the hydrolysis; subsequent mutarotation leads eventually to an equilibrium mixture of the α and β forms, in molar ratio 8:92. In a mixed water/methanol solution, the hydrolysis reaction produces also the α-methyl sialoside but not its β-methyl counterpart. We also show that 4-methyl-umbelliferyl-N-acetylneuraminic acid is a significantly better substrate for the sialidase than α(2-3)-sialyllactose. Prolonged incubation of α(2-3)-sialyllactose with an excess of trans-sialidase produced a trace of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, as identified by NMR spectroscopy and by gas liquid chromatography/mass spectrometry. In conclusion, this study shows that the stereoselectivity of the sialidase activity of T. cruzi trans-sialidase is identical to that of bacterial, viral, and mammalian sialidases, suggesting a similar active-site architecture.