Trans-sialidase from Trypanosoma cruzi catalyzes sialoside hydrolysis with retention of configuration.
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(1)H nuclear magnetic resonance (NMR) spectroscopy to investigate the stereospecificity of the hydrolysis of two substrates, namely, 4-methyl-umbelliferyl- N -acetylneur-aminic acid and alpha(2-3)-sialyllactose, catalyzed by a recombinant T.cruzi trans -sialidase. We demonstrate that, in aqueous solution, the thermodynamically less stable alpha-form of N -acetylneuraminic acid is the initial product of the hydrolysis; subsequent mutarotation leads eventually to an equilibrium mixture of the alpha and beta forms, in molar ratio 8:92. In a mixed water/methanol solution, the hydrolysis reaction produces also the alpha-methyl sialoside but not its beta-methyl counterpart. We also show that 4-methyl-umbelliferyl- N -acetylneuraminic acid is a significantly better substrate for the sialidase than alpha(2-3)-sialyllactose. Prolonged incubation of alpha(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 spectro-metry. In conclusion, this study shows that the stereo-selectivity 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.
Todeschini
A R
AR
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21944-970, Cidade Universitária, Rio de Janeiro, Brasil.
Mendonça-Previato
L
L
Previato
J O
JO
Varki
A
A
van Halbeek
H
H
D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism; D002236:Carbohydrate Conformation; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014349:Trypanosoma cruzi / Q000201:enzymology
D000953:Antigens, Protozoan; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; C069715:trans-sialidase; D009439:Neuraminidase