CMP-sialic acid synthase

The extensive manipulations which have been carried out on sialic acids themselves have been reviewed b) Tupp) and Gottschalk [63], Holmquist [101], Vliegenthart and Kammerling [102], in detail by Zbiral [4], and recently by von Itzstein and Kiefel [103]. This account will therefore give an overview of the transformations which have been carried out, resulting in modification at every carbon of NeuSAc. The derivatives prepared have been used to probe the requirements of sialic acid-recognising proteins, in particular the enzymes involved in sialic acid metabolism such as CMP-sialate synthase, sialidases, and NeuSAc 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-... 

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. 

The enzyme CMP-Neu5Ac synthase, which is also known as Neu5Ac cytidylyltransferase (EC 2.7.7.43), is ubiquitous in pro- and eukaryotic cells synthesizing sialic acids from glucose [5,33]. In contrast to other nucleotide-sugar-synthesizing enzymes, it is located... 

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 sialyltransferase assay is usually carried out with radioactive CMP-Neu5Ac and the sialic acid acceptor, followed by identification of the radioactive product. As this product is often membrane-bound and the radioactive cosubstrate is expensive, a non-radioactive assay was developed [625], in which CMP released from CMP-Neu5Ac by the transfer of Neu5Ac is determined with HPLC (see section 5.3.2). Since CMP-Neu5Ac can be analyzed in the same run, this test is also suited for the activity of the CMP-Neu5Ac synthase described above. 

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