Neu5Ac-synthetase

N. meningitides and human ST6Gal-I, EC2.4.99.1) were exploited to add sialic acid in a2-3 and a2-6 linkages to the terminal galactose residue of mono-, di- and tri-LacNAc. The fusion of the bacterial ST3 with CMP-Neu5Ac synthetase [83] generated the donor substrate in situ from added NeuSAc and CTP. 

A common practice is to conduct the immobilization procedure in the presence of species that occupy the active site of the enzymes, such as substrates, cofactors, reversible competitive inhibitors, or products, at concentrations preferably above their Michaelis or inhibition constants. Such a precaution is not necessary for the immobilization of CMP-Neu5Ac synthetase. 

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffuse through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffuse into the outside solution, where they may be recovered. This technique has been used in syntheses with sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2— 6) sialyl transferase,26 a mixture of pyruvate kinase and adenylate kinase,27 and CMP-Neu5Ac synthetase.28... 

A simplified one-pot two-step enzymatic approach was used by James Paulson s group to produce sialoside derivatives (23). Instead of recycling the CMP-sialic acid, the sialyltransferase donors with modifications at C-5 or C-9 position of NeuSAc were enzymatically synthesized from ManNAc or its C-2 or C-6 modified derivatives, pyruvate, and CTP using a sialic acid aldolase and a CMP-Neu5Ac synthetase/sialyltransferase fusion protein. After removing the protein by membrane filtration, the filtrate containing produced CMP-sialic acid... 

Figure 10 The sialoside biosynthetic pathway in eukaryotic cells A, UDP-GlcNAc-2-epimerase B, ManNAc-6-kinase C, Neu5Ac-9-P04 synthase D, NeuSAc-b-POj phosphatase E, CMP-Neu5Ac-synthetase F, sialyltransferase. Enzymes A and B function together as a bifunctional enzyme.

Sialic acid biosynthesis in bacteria is less complex than in mammals. There are only three enzymes involved in NeuSAc biosynthesis they are the NeuSAc-synthase, CMP-Neu5Ac-synthetase, and sialyltransferase. Although these enzymes were cloned only within the past 10 years, their straightforward overexpression in bacteria has allowed extensive studies to be performed. 

CMP-Neu5Ac Synthetase Origin E. coli K 235/CSl JQlich Enzyme Products... 

Synthesis of iV-acetylneuraminic acid (Neu5Ac) in vivo is catalyzed by Neu5Ac synthetase (EC 4.1.3.19) through the irreversible condensation of phosphoenolpyruvate (PEP) and A/-acetylmannosamine (15) (Scheme 2) [30-32], This enzyme has not yet been isolated and its catalytic activity might be interesting field for exploration. 

Figure 3. Enzymatic one-pot synthetic procedure of sialyloligosaccharides. A NeuSAc-aldolase, B CMP-Neu5Ac synthetase, C sialyltransferase (adaptedfrom...

The total synthesis of sialosides by using the chemoenzymatic approach is as follows [74]. Sialic acid itself can be synthesized from ManNAc, mannose, or their derivatives by sialic acid aldolase enzyme through aldol condensation reaction. If ManNAc is chemically or enzymatically modified at C2, C4—C6 positions, sialic acid has structural modifications at C5, C7-C9 positions, respectively. The sialic acids are subsequently activated by a CMP-siahc acid synthetase to form a CMP-sialic acid, which is the donor used by sialyltransferases. Because CMP-sialic acid is tmstable, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialosides. In the last steps, the CMP-sialic acid is transferred to galactose or GalNAc terminated glycosides by sialyltransferases to form structurally defined sialosides. Examples are that Chen and co-workers have recently developed a one-pot multienzyme system for the efficient synthesis of a-sialosides (Table 2) [12,76,79]. In this system, recombinant E. coli K-12 sialic acid aldolase catalyzed the synthesis of sialic acid precursors for... 

CMP-Neu5Ac synthetase cytidine-5 -triphosphate Inorganic pyrophosphatase... 

Several a3-sialyltransferases have been cloned, and ST3Gal III is commercially available in recombinant form. A particularly interesting report of Gilbert et al. [55] describe a fusion protein consisting of CMP-Neu5Ac synthetase and o2,3-sialyl-transferase from Neisseria meningitidis. This polypeptide was able to catalyze the reactions shown in eqs (2) and (3). 

---