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

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 Mn(2+). The enzyme is active at pH 8-13 in Tris-HCl buffer and at 37-60 degrees C, and maximum activity is observed at pH 9.5 and 50 degrees 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 microM, 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.