Enzyme Neu5Ac-aldolase

Free sialic acids are used by the enzymes of sialic acid metabolism, CMP-sialate synthase and Neu5Ac aldolase. However the majority of sialic acid-metabolising and -recognising proteins interact with glycosidically bound sialic acid. There-... 

In contrast, Neu5Ac aldolase turned out to be very useful for synthetic purpose because it tolerate wide range of unnatural substrates [21,23]. The enzyme also named sialic acid aldolase (EC 4.1.3.3.) catalyze reversible reaction of A/-acetyl-D-mannosamine (15) and pyruvate (Scheme 3) [33-35],... 

Neu5Ac aldolase has been isolated from many sources including bacteria and mammals. Significant interest toward sialic acid analogs and commercial availability of the aldolase, have led to the intensive studies on this enzyme, which resulted in the knowledge that despite its highly specificity for pyruvate [36] it accepts a number of different acceptor... 

A sequential aldol reaction leading to sialic acid derivatives has been also presented [59], Combination of acetaldehyde dependent aldolase (DERA) and Neu5Ac aldolase catalyzed reactions led to (R)-C-4 keto acids. In this case, however, one-pot reaction sequence was not possible due to the incompatibility of the reaction conditions for two enzymes. 

As Neu5Ac aldolase accepts a range of substrates (more than 60 are known) in place of ManNAc, this enzyme can also used to synthesize Neu5Ac derivatives [149]. For example, if ManNAc is replaced by D-mannose, Neu5Ac aldolase can be used for the preparative-scale synthesis of KDN (3-deoxy-(5-D-g/ycer0-D-g /acto-2-nonulosonic acid Fig. 7) [150],... 

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... 

Fig. 13. Metabolism scheme of sialic acids. Anabolic (solid arrow) and catabolic (dashed arrow) reactions are indicated. For literature see the text. Enzymes 1, CMP-sialate synthase (EC 2.7.7.43) 2, sialyltransferases (EC 2.4.99.1.) 3, CMP-Neu5Ac hydroxylase (EC 1.14.99.18) 4, acetyl-CoA sialate 4-0-acetyltransferase (EC 2.3.1.44) 5, acetyl-CoA sialate 7(9)-0-acetyltransferase (EC 2.3.1.45) 6,. S-adenosyl-L-methionine sialate 8-O-methyltransferase (proposed EC 2.1.1.78) 7, sialate 4- or 9-0-acetylesterases (EC 3.1.1.53) 8, sialidase (EC 3.2.1.18) 9, sialate-pyruvate lyase (aldolase EC 4.1.3.3). Both Neu5Ac and Neu5Gc can be O-acetylated by the two O-acetyltransferases. There may also exist a sulfotransferase, since sulfated sialic acids have been found in e.g. echinoderms [13,577]. ( ), Sialic-acid-accepting nascent glycoconjugate.

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... 

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