Pyruvate lyase

TV-Acetyl neuraminic acid aldolase [from Clostridium perfringens, TV-acetyIneuraminic acid pyruvate lyase] [9027-60-5] 32,000 [EC 4.1.3.3]. Purified by extraction with H2O,... 

Many other known pyruvate lyases are unsuited to application in asymmetric synthesis since they catalyze C-C bond formation with random stereochemistry30. 

Mechanistically similar to the pyruvate lyases, 2-deoxy-D-ribose 5-phosphate aldolase (EC 4.1.2.4) catalyzes the addition of acetaldehyde to D-glyceraldehyde 3-phosphate. 

This enzyme [EC 4.1.3.17], also known as 4-hydroxy-4-methyl-2-oxoglutarate aldolase and 4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase, catalyzes the reversible conversion of 4-hydroxy-4-methyl-2-oxoglutarate to pro-... 

Tentative localization of acetyl groups at the 0-4 atoms of sialic acids in native complex carbohydrates is possible with the aid of siali-dases, which are almost, or completely, inactive with these compounds or, after isolation of the sialic acids, by acylneuraminate pyruvate-lyase, which also shows little activity with these substrates (see Section VI). 

Thin-layer chromatography of 2-acylamido-2-deoxy-D-mannoses resulting from cleavage of sialic acids with acylneuraininate pyruvate-lyase is described in Ref. 107, together with the RF values of the different 0-acyl-2-acylamido-2-deoxy-D-mannose species. 

That secretion of 2-acetamidoglucal, which is known to be an intermediate in the complex, 2-epimerase reaction, but cannot be isolated under normal conditions, may point to another defect in this enzyme that may be independent of the presence or absence of a feedback-inhibition receptor site for CMP-Neu5Ac has been discussed.233 An excess of 2-acetamidoglucal may be converted spontaneously into GlcNAc and this, enzymically, into ManNAc both compounds are secreted in the urine of the sialuria patient. As a consequence of these reactions, the cellular concentration of ManNAc may increase to a level leading to the synthesis of additional Neu5Ac from this compound by the action of acylneuraminate pyruvate-lyase. All of these reactions, and known or theoretical interactions, were summarized in Fig. 3 of Ref. 233. 

Scheme 3.—Proposed Mechanism of Reversible Cleavage of Neu5Ac by Acylneur-aminate Pyruvate-lyase. (Taken, and modified, from Ref. 408.)...

Scheme 4.—Induction and Cooperation of the Sialic acid-specific Enzymes Siali-dase, Acylneuraminate Pyruvate-lyase, and Neu5Ac-permease (hypothetical) in Clostridium perfringens. [Key , sialoglycoprotein , free sialic acid f—at>, ...

On the basis of this anti-proteolytic effect of sialic acids, a hypothetical model435 for the role of sialidase in clostridial infections is shown in Scheme 4. It is considered that the bacterial enzyme releases sialic acids from cell-surface glycoproteins of the infected tissue, which thereafter can be readily attacked by proteases. This cooperation between sialidase and protease may support the spreading of the bacteria. Acylneuraminate pyruvate-lyase, also shown in this model, degrades sialic acids for energy supply, and growth, of the bacteria. 

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). 

C. Aug6, S. David, C. Gautlteron, A. Malleron, and B. Cavayd, Preparation of six naturally occurring sialic acids with immobilized acylneuraminate pyruvate lyase, New J. Chem. 72 733 (1988). 

Sialic acid aldolase (SA EC 4.1.3.3), also named A-acetylneuraminate pyruvate lyase, has been extensively used by our group in its immobilized form, first for the synthesis of large amounts of A-acety lneuraminic acid [20] and then for many natural and unnatural sialic acids [21], SA catalyzes the reversible aldol reaction of A-acetylmannosamine and pyruvate to give A-acety lneuraminic acid with an optimum pH for activity of 7.5 and an equilibrium constant of 12.7 A/-1 in the synthetic direction (Scheme 3) [10],... 

C. Aug6, B. Bouxom, B. Cavayd, and C. Gautheron, Scope and limitations of the aldol condensation catalyzed by immobilized acylneuraminate pyruvate lyase, Tetrahedron Lett. 30 2217 (1989). 

In vivo, pyruvate lyases perform a catabolic function. The synthetically most interesting types are those involved in the degradation of sialic acids or the structurally related octulosonic acid KDO, which are higher sugars typically found in mammalian or bacterial glycoconjugates [62-64], respectively. Also, hexose or pentose catabolism may proceed via pyruvate cleavage from intermediate 2-keto-3-deoxy derivatives which result from dehydration of the corresponding aldonic acids. Since these aldol additions are freely reversible, the often unfavourable equilibrium constants require that reactions in the direction of synthesis have to be driven by an excess of one of the components, preferably pyruvate for economic reasons, in order to achieve a satisfactory conversion. 

Comparable to the situation for the sialic acid and KDO lyases (vide supra), sets of stereochemically complementary pyruvate lyases are known, e,g. in Pseudomonas strains, which act on related 2-keto-3-deoxy-aldonic acids [112]. The enzymes cleaving six-carbon sugar acid phosphates—the KdgA and 2-keto-3-deoxy-6-phospho-D-galactonate (20) aldolases (KDPGal aldolase EC 4.1.2.21) [139] — are typified as class I enzymes, whereas those acting on non-phosphorylated five-carbon substrates — 2-keto-3-deoxy-L-arabonate (21) (KDAra aldolase EC 4.1.2,18) [140, 141] and 2-keto-3-deoxy-D-xylonate (22)... 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

Deoxy-D-manno-octulosonate 8-phosphate synthetase /V-Acetylneuraminate pyruvate lyase... 

Condensations with pyruvate in the presence of V-acylneuraminate pyruvate lyase immobilized on agarose. 

Figure 3-5. Biosynthesis of salicylic acid. The enzymes involved in this pathway are (a) chorismate mutase (E.C. 5.4.99.5), (b) prephenate aminotransferase (E.C. 2.6.1.78 and E.C. 2.6.1.79), (c) arogenate dehydratase (E.C. 4.2.1.91), (d) phenylalanine ammonia lyase (E.C. 4.3.1.5), (e) presumed P-oxidation by a yet to be identified enzyme, (f) benzoic acid 2-hydroxylase, (g) isochorismate synthase (E. C. 5.4.4.2), and (h) a putative plant pyruvate lyase.

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