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.
Mizanur
Rahman M
RM
Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames, IA, 50011, USA. npohl@iastate.edu
Pohl
Nicola L
NL
D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D008970:Molecular Weight; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
D004269:DNA, Bacterial; D000216:N-Acylneuraminate Cytidylyltransferase