Aldolase organ-specific

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

The Schiff-base-forming types (class I) are known only for the two former aldolases (FruA, TagA), which are found usually in mammalian or (as an exception) in specific microbial organisms, whereas the Zn2+-dependent type (class II) comprises all four DHAP aldolases which are commonly found in bacteria [43], Typically, type I FruA enzymes are tetrameric proteins composed of subunits of 40 kDa [191,192], while the type II FruA are dimers of 39 kDa subunits [193]. RhuA and FucA enzymes are homotetrameric with a subunit molecular weight of 25 kDa and 30 kDa respectively [194,195],... 

To date, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) is the only acetaldehyde-dependent aldolase being applied in organic synthesis. Thus the stereoselectivity of DERA is significant, all known enzymes from different organisms showing the same preferences, limiting the field of application to syntheses in which specifically the DERA-catalyzed enantiomer is needed. 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

The amino acid sequence results clearly Imply a unique sequence for each of the enzymes examined, and there is no decisive evidence for the existence among GAPDH s of tissue-specific isozymes that differ in primary sequence despite reports of the occurrence of multiple electrophoretic forms in several different organisms (40, 41)- In no case was it demonstrated that these multiple forms are the products of different genes, and it is entirely possible that electrophoretically different tetra-mers may have arisen by amide loss [as in the case of muscle aldolases (4 ) ] or through differential binding of NAD (41). 

Izzo P, Costanzo P, Lupo A, Rippa E, Paolella G, Salvatore F. Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing. Eur J Biochem 1988 174 569-78. 

There have been more than 20 aldolases isolated, eight of which have been explored for organic synthesis (6). Aldolases possess two interesting common features the enzymes are specific for the donor substrate but flexible for the acceptor component, and the stereochemistry of aldol reaction is controlled by the enzyme not by the substrates. In our previous study, we have described the use of lipases, hexokinases, glycosyl transferases and rabbit muscle aldolase for the synthesis of certain fluorosugars (7). This review describes our recent development in aldolase-catalyzed reactions for the synthesis of fluorosugars. 

Several characteristics of FDP aldolase make it a useful enzyme for use in synthesis. Commercial preparations of the enzyme are inexpensive (it costs 0.04 to produce 1 mmol of product per minute, i.e. 0.04 unir and it has a reasonable specific activity (60 unit mg" of protein). FDP aldolase requires no metal ions or cofactors and it is stable in the presence of oxygen and added organic cosolvents, and is not... 

A large number of other aldolases have been isolated and characterized (Table 5).33.42.43.i 11-127 Limited explorations of substrate specificity have been made in many cases but these enzymes have not yet been used in synthetic organic chemistry. In terms of potential utility as catalysts, the aldolases of bacterial origin may be of more use than the enzymes from plant or animal sources because the former are more easily cloned and altered by genetic engineering than the latter. The alterations should prove to be useful for controlling the substrate specificity of these enzymes. 

A final example of an aldolase-catalysed reaction is that between D-glyceraldehyde-3-phosphate (1) (electrophile) and acetaldehyde (nucleophile) (Scheme 5.10). It is interesting to note that the enzyme carefully organizes the reactants such that acetaldehyde acts only as the nucleophile component of the reactant, but not the electrophile. This aldolase has been shown to feature relaxed specificity with respect to both of the substrates. 

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