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Cloning and characterization of a heat-stable CMP-N-acylneuraminic acid synthetase from Clostridium thermocellum

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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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Acknowledgements

We thank a Shimadzu University Research Grant and the Herman Frasch Foundation (American Chemical Society) for their support of this research. N.L.P. is a Cottrell Scholar of Research Corporation and an Alfred P. Sloan Foundation Fellow.

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  1. Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames, IA, 50011, USA

    Rahman M. Mizanur & Nicola L. Pohl

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  1. Rahman M. Mizanur
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  2. Nicola L. Pohl
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Correspondence to Nicola L. Pohl.

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Mizanur, R.M., Pohl, N.L. Cloning and characterization of a heat-stable CMP-N-acylneuraminic acid synthetase from Clostridium thermocellum . Appl Microbiol Biotechnol 76, 827–834 (2007). https://doi.org/10.1007/s00253-007-1053-2

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  • DOI: https://doi.org/10.1007/s00253-007-1053-2

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