Acetylneuraminic acid phosphate

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.

The N-acetylneuraminic acid derivative 44 is widely distributed. It was isolated from a strain182 of Escherichia coli, and has been obtained from cytidine 5 -triphosphate and N-acetylneuraminic acid by the action of enzyme preparations from Neisseria meningitidis183 and from animal tissues.184-186 The latter enzyme can also make use of N-glycolylneuraminic acid as a substrate, to give the respective cytidine 5 -phosphate derivative. 

The structure shown was confirmed by the identification of cytidine 5 -phosphate and N-acetylneuraminic acid after mild acidic hydrolysis, and by the stability of the derivative on treatment with sodium borohydride. Comparison of the optical rotation of the starting derivative182 (44), the products of its hydrolysis,182 and the anomeric... 

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... 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

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. 

Two of the most frequent monosaccharide components of bacterial polymers belonging to this group have been the subjects of articles in this Series. They are 3-deoxy-D-manno-2-octulosonic acid,247 a normal constituent of the core region of bacterial lipopolysaccharides that is also present in some other polymers, and N-acetylneuraminic acid,248 found in several capsular polysaccharides. Enolpyruvate phosphate serves as the precursor of the C-l-C-3 fragment of the monosaccharides, with D-arabinose 5-phosphate or 2-acetamido-2-deoxy-D-mannose 6-phosphate being an acceptor for transfer of the three-carbon unit. Characteristic, activated forms of these monosaccharides are the CMP derivatives. 

UTP a-D-glucose 1-phosphate uridyltransferase CMP-acetylneuraminic acid synthetase... 

Immobilized sialyl aldolase (50 mL of gel, 68 U) was added to a mixture of 88% pure A-acetylmannosamine (20 mmol), sodium pyruvate (180 mmol), 1,4-dithiothreitol (0.2 mmol), and sodium azide (20 mg) in 0.05 M potassium phosphate buffer, pH 7 (150 mL). The suspension was gently stirred under nitrogen for 4 d at 37°, the reaction being monitored by t.l.c. in 7 3 propanol - water. The gel was removed by filtration, washed with the buffer, and A-acetylneuraminic acid (2) was isolated by chromatography on Dowex 1 X8 (HCOj-) resin, using a gradient of formic acid as the eluant, in 66% yield. The gel was used in four successive runs. Starting from 17 g of 88% pure A-acetylmannosamine, the procedure allowed the synthesis of 14 g of A-acetylneuraminic acid (2). In the end, the recovered gel retained 80% of its enzymic activity. 

The N-acetylneuraminic acid in the RBC membrane can be removed by incubating RBCs with neuraminidase. Incubation of a dilute human RBC suspension (1% by volume) at 37°C for 1 hr with 15 /xg/mL of neuraminidase causes the complete removal of N-acetylneuraminic acid and a reduction of surface potential by 90% (24). The residual negative surface charges can be attributed to the phosphate groups of the membrane phospholipids or the carboxyl groups of the membrane proteins (28). 

CMP-KDO synthetase (cytidine-5 -triphosphate cytidine-5 -mon-ophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase), the next enzyme in the pathway, catalyzes the formation of the nucleotide sugar, CMP-KDO from CTP and KDO. This enzyme was first studied by Ghalambor and Heath (IT). We have purified this enzyme to homogeneity (27). T i apparent K values for CTP nd KDO in the presence of 10 mM Mg were determined to be 2 x 10 M and 2.9 x 10 M, respectively. The enzy tic reaction was dependent upon the addition of CTP, KDO and Mg but did not require a reducing agent. The formation of CMP-KDO was not inhibited by the addition of CDP, CMP, KDO-8-phosphate or N-acetylneuraminic acid to the complete reaction mixture. In agreement with Ghalambor and Health (17), neither KDO-8-phosphate nor N-acetylneuraminic acid could substitute for KDO in the reaction mixture. Pyrophosphate, one of the end products, is a weak inhibitor of the reaction with an apparent Ijq value of 5.0 mM. The addition of CMP,CD or any of the other mono- or di-nucleotides did not inhibit the reaction. 

UDP-D-xylose, GDP-D-mannose, GDP-L-Fucose, or CMP-D-A-acetylneuraminic acid. Mammalian GTs also use dolichol-diphosphate-GlcNAc2Man9GlC3, dolichol-phosphate-mannose and dolichol-phosphate-glucose (Fig. 2). 

Figure 6-15. Synthesis of sphingolipids. NANA = W-acetylneuraminic acid G/c = glucose Gal = galactose GalNAc = N-acetylgalactosamine PIP = pyridoxal phosphate FA = fatty acyl groups derived from fatty acids = hydrophobic chains of ceramide. The dashed box contains the portion of ceramide derived from serine.

In contrast to other monosaccharides, activated sialic acid donors are biosynthesized from A -acetylmannosamine (ManNAc) or directly from sialic acids (Sia), including A-acetylneuraminic acid (NeuAc), via a more complex pathway (21). ManNAc is phosphorylated at the at the 6-hydroxyl group and condensed with phosphoenolpyruvate to give A -acetylneuraminic acid-9-phosphate (NeuAc-9-P). Phosphate ester hydrolysis is followed by direct condensation with CTP to give CMP-NeuAc (Figure 3). Sialic acids can intercept this pathway directly via enzymatic reaction with CTP. 

N-acetylneuraminic acid N-glycolylneuraminic acid palmitoyl-CoA sphingosine 1-phosphate sphinganine 1-phosphate sphingolipid-activator protein serine... 

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