Cloning and characterization of a heat-stable CMP-<Emphasis Type="Italic">N</Emphasis>-acylneuraminic acid synthetase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> |
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Authors: | Rahman M Mizanur Nicola L Pohl |
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Institution: | (1) Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames, IA 50011, USA |
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Abstract: | In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme
has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn2+. The enzyme is active at pH 8–13 in Tris–HCl buffer and at 37–60 °C, and maximum activity is observed at pH 9.5 and 50 °C
in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc
are 240 ± 20, 130 ± 10, and 160 ± 10 μM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s−1, respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi–bi catalytic mechanism. In addition
to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc
synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms. |
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