Sialic acid synthase

Merker, R. I., and Troy, F. A., 1990, Biosynthesis of the polysialic acid capsule in Escherichia coli Kl. Cold inactivation of sialic acid synthase regulates capsule expression below 20°C, Gly-cobiology 1 93-100. 

Synthetic studies for sialic acid and its modifications have extensively used the catabolic enzyme N-acetylneuraminic acid aldolase (NeuA E.C. 4.1.3.3), which catalyzes the reversible addition of pyruvate (70) to N-acetyl-D-mannosamine (ManNAc, 11) to form the parent sialic acid N-acetylneuraminic acid (NeuSNAc, 12 Scheme 2.2.5.23) [1, 2, 45]. In contrast, the N-acetylneuraminic acid synthase (NeuS E.C. 4.1.3.19) has practically been ignored, although it holds considerable synthetic potential in that the enzyme utilizes phosphoenolpyruvate (PEP, 71) as a preformed enol nucleophile from which release of inorganic phosphate during... 

Scheme 2.2.5.23 Alternative pathways for sialic acid synthesis using the catabolic aldolase (NeuA) or the anabolic synthase (NeuS) enzymes.

The availability of both the cataboHc aldolase and the uniquely synthetic anabolic synthase made it possible to assemble a novel continuous assay for the determination of the metabolite N-acetylneuraminic acid [46]. A combination of both enzymes, in the presence of an excess of PEP, will start a cycle in which the determinant sialic acid will undergo a steady conversion of cleavage and re-syn-thesis as a futile cycle (Scheme 2.2.5.24). With each progression, however, 1 equiv of pyruvate is liberated simultaneously, which causes time-dependent signal amplification. Pyruvate is quantified spectrophotometrically by a corresponding NADH consumption when the system is coupled to the standard pyruvate dehy-... 

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. 

It was not imtil 4 years ago that the first bacterial NeuSAc-synthase was cloned [136]. This enzyme, derived from E. coli Kl, is imlike the mammalian enzyme in that it does not accept the ManNAc-b-POj as a substrate. Rather, it condenses ManNAc with phosphoenolpyruvate (PEP) to produce NeuSAc. Fmthermore, 6-N3 ManNAc can be converted to 9-Na NeuSAc, although only with S9% efficiency compared to the native substrate [137]. The E. coli synthase has also been shown to accept ManN as a substrate, albeit with oidy a miiumal production of the sialic acid... 

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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