Sialic acid 9-phosphate

Watson, D. R., Jordian, G. W., and Roseman, S. The sialic acids Sialic acid 9-phosphate synthetase. J. Biol Chem., 1966, 241, 5627-5636. 

Roseman, S., Jourdian, G. W., Watson, D., and Rood, R., 1961, Enzymatic synthesis of sialic acid-9-phosphate, Proc. Natl. Acad. Sci. USA 47 958-961. 

Unlike the previous enzyme, NAN-aldolase, this enzyme catalyzes an essentially irreversible reaction in favor of synthesis. The enzyme was first found in rat liver extracts (Warren and Felsenfeld, 96 a) and has been purified some 95-fold from bovine submaxillary gland (Watson et aL, 1966). It can utilize both A -acetyl as well as N-glycolylmannosamine-6-P04. It seems likely that this enzyme, sialic acid-9-phosphate synthetase, catalyzes the major reaction for the synthesis of sialic acid. The product of the reaction is the 9-phosphate ester of sialic acid and must be dephosphorylated before activation can occur. 

A number of nonspecific phosphatases can act on NAN-9-PO4 to dephosphorylate it. However, it appears that in rat liver there is a specific sialic acid-9-phosphatase (Warren and Felsenfeld, 1962). A similar enzyme has been purified some 800-fold from human erythrocyte lysates (Jourdian et al., 1964) and has been shown to be highly specific for sialic acid 9-phosphate (both A/ -acetyl and A/ -glycolyl). This enzyme completes the synthesis of sialic acid in animal tissues. In bacteria, however, another pathway does exist and may be widespread. Extracts of Neisseria meningitidis synthesize sialic acid utilizing free N-acetyl-... 

In summary, there are three known pathways for the synthesis of sialic acid the NAN-aldolase, which condenses pyruvate and N-acylmannosamine (bacterial and animal) sialic acid-9-phosphate synthetase, which condenses phosphoenolpyruvate with AT-acylmannosa-mine-6-phosphate (animal tissue only) the enzyme from Neisseria meningitidis, which condenses N-acylmannosamine with phosphoenolpyruvate (bacteria only). 

As 2-amino-2-deoxy-D-mannose is tumorstatic and 2-acetamido-2-deoxy-D-mannose 6-phosphate is an obligatory intermediate in the biosynthetic pathway to sialic acid, displacement of the essential OH-6 with a fluorine atom should be interesting from the biological viewpoint. 2-Acetamido-1,3,4-tri-0-acetyl-2,6-dideoxy-6-fluoro-D-mannopyranose (see Table 111 in Section 11,3) and its O- and A,0-deacetyl derivatives were prepared the first compound showed weak anticancer activity. 

Add 106-108cells/ml in a PBS solution (lOmM sodium phosphate, 0.15M NaCl, pFI 7.4) containing ImM sodium periodate and incubate on ice for 30 minutes in the dark. This level of periodate addition will target the oxidation only to sialic acid residues (Chapter 1, Section 4.4). If additional sites of glycosylation also are to be labeled, increase the periodate concentration to 10 mM and do the reaction at room temperature in the dark. 

Dissolve the glycoprotein(s) to be labeled in ice-cold ImM sodium periodate, lOmM sodium phosphate, 0.15 M NaCl, pH 7.4, for the exclusive oxidation of sialic acid residues. For general carbohydrate oxidation, increase the periodate concentration to 10 mM in PBS at room temperature. 

Dissolve a glycoprotein to be labeled in 50mM sodium phosphate, pH 7 (reaction buffer), at a concentration of l-10mg/ml. For sialic acid modification, place the sample in ice to cool to near 0°C. 

Of the known classes of aldolase, DERA (statin side chain) and pyruvate aldolases (sialic acids) have been shown to be of particular value in API production as they use readily accessible substrates. Glycine-dependent aldolases are another valuable class that allow access to p-hydroxy amino acid derivatives. In contrast, dihydroxy acetone phosphate (DHAP) aldolases, which also access two stereogenic centres simultaneously,... 

Figure 2.19 Sialylation ofN-acetyl lactose by cytidyl monophosphate-N-acetylneuraminic acid using Of 2,3-neuraminic acid transferase as catalyst (upper box). Regeneration of the sugar nucleotide is shown in the lower box. CMP is converted into CTP in two steps using two different kinases. In the final step CMP-A -acetylneuraminic acid is synthesised from CTP and A -acetylneuraminic acid (sialic acid) using the appropriate synthetase. The formed pyrophosphate is converted into inorganic phosphate. Altogether five different enzymes are involved in the process.

Some bacteria uhlize phosphoenolpyruvate (PEP 71)-dependent lyases for the generation of sialic acids and related compounds (Scheme 2.2.5.4) [1]. By simultaneous release of inorganic phosphate from the preformed enolpyruvate nucleo-... 

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... 

The occurrence of phosphoric ester groups at 0-9 is long established, as Neu5Ac9P was recognized as the condensation product of enolpyruvate phosphate (PEP) and ManNAc 6-phosphate in the biosynthetic pathway of sialic acids (see Section V,l). 

Fig. 4.3.2 Human sialic acid metabolism and genetic defects. -6P -6-Phosphate, -9P -9-phos-phate, CMP cytidine 5 -monophosphate, CTP cytidine 5 -triphosphate, UDP-GlcNAc uridine diphosphate-N-acetyl-D-glucosamine, ManNAc N-acetylmannosamine, NeuAc N-acetylneur-aminic acid, OGS oligosaccharides, SASD sialic acid storage disease...

Properties and Structure. a1-Acid glycoprotein (a1-AGP) has a molecular mass of about 41,000 and consists of a peptide chain having 181 amino acid residues and five carbohydrate units (14,15). Two cystine disulfide cross-linkages connect residues 5 and 147 and residues 72 and 164. The carbohydrate units comprise 45% of the molecule and contain sialic acid, hexosamine, and neutral hexoses. In phosphate buffer the isoelectric point of the... 

Eupergit 250L (Rohm Chemie, 400 mg) was added to a solution of sialic acid aldolase (8 mg, 64 U) in 1 M potassium phosphate buffer pH 7.4 (3.2 mL) containing 0.04 M sodium pyruvate and 0.02% NaN3 the suspension was stirred for 3 days at room temperature under N2. The gel was washed with 0.1 M potassium phosphate buffer pH 7 (10 mL) and stored at 4°C in this buffer in the presence of 0.04 M pyruvate and 10 3 M dithiothreitol. A unit yield of 40% was usually obtained for immobilization. 

Shikimate 3-phosphate 687s SH2 / phosphopeptide complex 368s Sialic acid 165s, 183, 546 Sialidase 186... 

The six-carbon chain of ManNAc 6-P can be extended by three carbon atoms using an aldol-type condensation with a three-carbon fragment from PEP (Eq. 20-7, step c) to give N-acetylneuraminic acid (sialic acid).48 Tire nine-carbon chain of this molecule can cyclize to form a pair of anomers with 6-membered rings as shown in Eq. 20-7. In a similar manner, arabi-nose 5-P is converted to the 8-carbon 3-deoxy-D-manno-octulosonic acid (KDO) (Fig. 4-15), a component of the lipopolysaccharide of gram-negative bacteria (Fig. 8-30), and D-Erythrose 4-P is converted to 3-deoxy-D-arafrmo-heptulosonate 7-P, the first metabolite in the shikimate pathway of aromatic synthesis (Fig. 25-1).48a The arabinose-P used for KDO synthesis is formed by isomerization of D-ribulose 5-P from the pentose phosphate pathway, and erythrose 4-P arises from the same pathway. 

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