Applied Microbiology and Biotechnology

, Volume 76, Issue 4, pp 827–834 | Cite as

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

Biotechnologically Relevant Enzymes and Proteins


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.


  1. Angata T, Varki A (2002) Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 102:439–469CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Bravo IG, Barrallo S, Ferrero MA, Rodriguez-Aparicio LB, Martinez-Blanco H, Reglero A (2001) Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2. Biochem J 358:585–598Google Scholar
  4. Bravo IG, Garcia-Vallve S, Romeu A, Reglero A (2004) Prokaryotic origin of cytidylyltransferases and alpha-ketoacid synthases. Trends Microbiol 12:120–127CrossRefGoogle Scholar
  5. Canganella F, Wiegel J (1993) The potential of thermophilic clostridia in biotechnology. In: Wood DR (ed) The clostridia and biotechnology. Butterworth-Heinemann, Boston, MA, pp 393–429Google Scholar
  6. Chokhawala HA, Yu H, Chen X (2007) High-throughput substrate specificity studies of sialidases by using chemoenzymatically synthesized sialoside libraries. ChemBioChem 8:194–201CrossRefGoogle Scholar
  7. Demian AL, Newcomb M, Wu JHD (2005) Cellulose, clostridia, and ethanol. Microbiol Mol Biol Rev 69:124–154CrossRefGoogle Scholar
  8. Gilbert M, Bayer R, Cunningham AM, DeFrees S, Gao Y, Watson DC, Young NM, Wakarchuk WW (1998) The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion. Nat Biotechnol 16:769–772CrossRefGoogle Scholar
  9. Haft RF, Wessels MR, Mebane MF, Conaty N, Rubens CE (1996) Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli. Mol Microbiol 19:555–563CrossRefGoogle Scholar
  10. Ishige K, Hamamoto T, Shiba T, Noguchi T (2001) Novel method for enzymatic synthesis of CMP-NeuAc. Biosci Biotechnol Biochem 65:1736–1740CrossRefGoogle Scholar
  11. Kean EL (1991) Sialic acid activation. Glycobiology 1:441–447CrossRefGoogle Scholar
  12. Kittelmann M, Klein T, Kragl U, Wandrey C, Ghisalba O (1995) CMP-N-acetyl neuraminic acid synthetase from Escherichia coli: fermentation production and application for the preparative synthesis of CMP-neuraminic acid. Appl Microbiol Biotechnol 44:59–67CrossRefGoogle Scholar
  13. Knorst M, Fessner W-D (2001) CMP-sialate synthetase from Neisseria meningitidis-overexpression and application to the synthesis of oligosaccharides containing modified sialic acids. Adv Synth Catal 343:698–710CrossRefGoogle Scholar
  14. Liu JL-C, Shen C-J, Ichikawa Y, Rutan JF, Zapata C, Vann WF, Wong C-H (1992) Overproduction of CMP-sialic acid synthetase for organic synthesis. J Am Chem Soc 114:3901–3910CrossRefGoogle Scholar
  15. Martinez J, Steenbergen S, Vimr E (1995) Derived structure of the putative sialic acid transporter from Escherichia coli predicts a novel sugar permease domain. J Bacteriol 177:6005–6010Google Scholar
  16. Mizanur RM, Zea CJ, Pohl NL (2004) Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides. J Am Chem Soc 126:15993–15998CrossRefGoogle Scholar
  17. Mizanur RM, Jaipuri FA, Pohl NL (2005) One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein. J Am Chem Soc 127:836–837CrossRefGoogle Scholar
  18. Mosimann SC, Gilbert M, Dombroswki D, To R, Wakarchuk W, Strynadka NCJ (2001) Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP. J Biol Chem 276:8190–8196CrossRefGoogle Scholar
  19. Plumbridge J, Vimr E (1999) Convergent pathways for utilization of the amino sugars N-acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli. J Bacteriol 181:47–54Google Scholar
  20. Rodriguez-Aparicio LB, Luengo JM, Gonzalez-Clemente C, Reglero A (1992) Purification and characterization of the nuclear cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from rat liver. J Biol Chem 267:9257–9263Google Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview, NYGoogle Scholar
  22. Samuels NM, Gibson BW, Miller SM (1999) Investigation of the kinetic mechanism of cytidine 5′monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate limiting steps from product inhibition analysis. Biochemistry 38:6195–6203CrossRefGoogle Scholar
  23. Schauer R (2000) Achievements and challenges of sialic acid research. Glycoconj J 17:485–499CrossRefGoogle Scholar
  24. Shames SL, Simon ES, Christopher CW, Schmid W, Whitesides GM, Yang LL (1991) CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N- acetylneuraminic acid and CMP-neuraminic acid derivatives. Glycobiology 1:187–191CrossRefGoogle Scholar
  25. Tullius MV, Munson Jr RS, Wang J, Gibson BW (1996) Purification, cloning, and expression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi. J Biol Chem 271:15373–15380CrossRefGoogle Scholar
  26. Tullius MV, Vann WF, Gibson BW (1999) Covalent modification of Lys19 in the CTP binding site of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase. Protein Sci 8:666–675CrossRefGoogle Scholar
  27. Vimr ER, Troy FA (1985) Identification of an inducible catabolic system for sialic acids (Nan) in Escherichia coli. J Bacteriol 164:845–853Google Scholar
  28. Vimr E, Lichtensteiger L (2002) To sialylate, or not to sialylate: that is the question. Trends Microbiol 10:254–257CrossRefGoogle Scholar
  29. Vimr E, Lichtensteiger C, Steenbergen S (2000) Sialic acid metabolism’s dual function in Haemophilus influenzae. Mol Microbiol 36:1113–1123CrossRefGoogle Scholar
  30. Warren L, Blacklow RS (1962) The biosynthesis of cytidine 5′-monophosphate-N-acetylneuraminic acid by an enzyme from N. meningitidis. J Biol Chem 237:3527–3534Google Scholar
  31. Yu H, Chokhawala H, Karpel R, Yu H, Wu B, Zhang J, Zhang Y, Jia Q, Chen X (2005) A multifunctional Pasteurella multocida sialyltransferase: a powerful tool for the synthesis of sialoside libraries. J Am Chem Soc 127:17618–17619CrossRefGoogle Scholar
  32. Yu H, Huang S, Chokhawala H, Sun M, Zheng H, Chen X (2006) Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural α-2,6-linked sialosides: a P. damsella α-2,6-sialyltransferase with extremely flexible donor-substrate specificity. Angew Chem Int Ed 45:3938–3944CrossRefGoogle Scholar
  33. Yu H, Yu H, Karpel R, Chen X (2004) Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases. Bioorg Med Chem 12:6427–6435CrossRefGoogle Scholar
  34. Zea CJ, Pohl NL (2004) General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry. Anal Biochem 328:196–202CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Chemistry and The Plant Sciences InstituteIowa State UniversityAmesUSA

Personalised recommendations