Aldolase substrate classes

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysiae S-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily ia microorganisms and utilize a divalent ziac to activate the electrophilic component of the reaction. The most studied aldolases are fmctose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn " -containing aldolase from E. coli. In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetone phosphate [57-04-5] (DHAP). 

Two classes of aldolase enzymes are found in nature. Animal tissues produce a Class I aldolase, characterized by the formation of a covalent Schiff base intermediate between an active-site lysine and the carbonyl group of the substrate. Class I aldolases do not require a divalent metal ion (and thus are not inhibited by EDTA) but are inhibited by sodium borohydride, NaBH4, in the presence of substrate (see A Deeper Look, page 622). Class II aldolases are produced mainly in bacteria and fungi and are not inhibited by borohydride, but do contain an active-site metal (normally zinc, Zn ) and are inhibited by EDTA. Cyanobacteria and some other simple organisms possess both classes of aldolase. 

Furthermore, the GPO procedure can also be used for a preparative synthesis of the corresponding phosphorothioate (37), phosphoramidate (38), and methylene phosphonate (39) analogs of (25) (Figure 10.20) from suitable diol precursors [106] to be used as aldolase substrates [102]. In fact, such isosteric replacements of the phosphate ester oxygen were found to be tolerable by a number of class I and class II aldolases, and only some specific enzymes failed to accept the less polar phosphonate (39) [107]. Thus, sugar phosphonates (e.g. (71)/(72)) that mimic metabolic intermediates but are hydrolytically stable to phosphatase degradation can be rapidly synthesized (Figure 10.28). 

It was reported by Horecker and coworkers that one class of aldolases (called Class I to distinguish it from the Class II aldolase that is metal ion-dependent) could be inhibited by the addition of borohydride reducing agent to reaction mixtures containing both enzyme and substrate129,130. It was then shown for the fructose- 1,6-bis-phosphate aldolase that the inhibition resulted from reduction of the Schiff base formed between the dihydroxyacetone phosphate substrate and the -amino group of a lysine side chain, thereby compromising the ability of the lysine to participate in subsequent turnover. 

Gefflaut, T., Blonski, C., Perie, J., and Willson, M., Class I aldolases Substrate specificity, mechanism, inhibitors and structural aspects. Prog. Biophys. Mol. Biol. 1995,63 (3), 301-340. 

Coincon, M., Wang, W., Sygusch, J., and Seah, S. Y. K., Crystal structure of reaction intermediates in pyruvate class II aldolase Substrate cleavage, enolate stabilization, and substrate specificity. /. Biol. Chem. 2012,287 (43), 36208-36221. 

Figure 1 Substrate classes of preparatively useful aldolases.

FIGURE 19.13 (a) A mechanism for the fructose-l,6-bisphosphate aldolase reaction. The Schlff base formed between the substrate carbonyl and an active-site lysine acts as an electron sink, Increasing the acidity of the /3-hydroxyl group and facilitating cleavage as shown. (B) In class II aldolases, an active-site Zn stabilizes the enolate Intermediate, leading to polarization of the substrate carbonyl group. 

Fructose bisphosphate aldolase of animal muscle is a Class I aldolase, which forms a Schiff base or imme intermediate between the substrate (fructose-1,6-bisP or dihydroxyacetone-P) and a lysine amino group at the enzyme active site. The chemical evidence for this intermediate comes from studies with the aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of fructose bisphosphate aldolase with dihydroxyacetone-P and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. 

These observations are explained by the mechanism shown in the figure. NaBH4 inactivates Class I aldolases by transfer of a hydride ion (H ) to the imine carbon atom of the enzyme-substrate adduct. The resulting secondary amine is stable to hydrolysis, and the active-site lysine is thus permanently modified and inactivated. NaBH4 inactivates Class I aldolases in the presence of either dihydroxyacetone-P or fructose-1,6-bisP, but inhibition doesn t occur in the presence of glyceraldehyde-3-P. 

The class I FruA isolated from rabbit muscle aldolase (RAMA) is the aldolase employed for preparative synthesis in the widest sense, owing to its commercial availability and useful specific activity of 20 U mg . Its operative stability in solution is limiting, but the more robust homologous enzyme from Staphylococcus carnosus has been cloned for overexpression [87], which offers unusual stability for synthetic purposes. Recently, it was shown that less polar substrates may be converted as highly concentrated water-in-oil emulsions [88]. 

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,... 

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). 

Aldolases have been classified into mechanistically distinct classes according to their mode of donor activation. Class 1 aldolases achieve stereospecific deprotonation via covalent imine/enamine formation at an active-site lysine residue, while Class II aldolases utilize a divalent transition metal cation for substrate coordination as an essential Lewis acid cofactor (usually Zn ) to facilitate deprotonation... 

II]. This latter feature facilitates racemate resolutions and allows the concurrent determination of three contiguous chiral centers in final products, which are obtained enantiopure and with high d.e. (>95) even when starting from more readily accessible racemic material. For certain substrates, however, diastereoselectivity of Class II aldolases can be compromised in the control of the stereocenter at C4, which points to occasional inverse binding of the respective aldehyde carbonyl [1,... 

In the catalysis of the lyase from C. perfringens, the participation of lysine residues forming intennediary Schiff bases between enzyme and substrate molecules, and of histidine residues, has been demonstrated with the aid of photooxidation, reagents for histidine modification, and borohydride reduction in the presence of substrate.408-418 Thus, according to Frazi and coworkers,414 the lyase belongs to the class I lyases (aldolases). The catalytic mechanism proposed is outlined in Scheme 3. Evidence has been educed for the existence of a similar mechanism of cleavage of sialic acid by the lyase enriched from pig kidney.411... 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

The most important chemical function of Zn2+ in enzymes is probably that of a Lewis acid providing a concentrated center of positive charge at a nucleophilic site on the substrate/ This role for Zn2+ is discussed for carboxypeptidases (Fig.12-16) and thermolysin, alkaline phosphatase (Fig. 12-23),h RNA polymerases, DNA polymerases, carbonic anhydrase (Fig. 13-1),1 class II aldolases (Fig. 13-7), some alcohol dehydrogenases (Fig. 15-5), and superoxide dismutases (Fig.16-22). Zinc ions in enzymes can often be replaced by Mn2+, Co2+, and other ions with substantial retention of catalytic activity/ ... 

Fig. 1. Mechanistic distinction of aldolases according to Schiff-base (class I) or metal-ion (class II) activation of substrates...

Fig. 2. Schematic representation of substrate binding and C-C bond formation for the class I fructose 1,6-bisphosphate aldolase from rabbit muscle...

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). 

Antibody Catalysis. Recent advances in biocatalysis have led to the generation of catalytic antibodies exhibiting aldolase activity by Lemer and Barbas. The antibody-catalyzed aldol addition reactions display remarkable enantioselectivity and substrate scope [18]. The requisite antibodies were produced through the process of reactive immunization wherein antibodies were raised against a [Tdiketone hapten. During the selection process, the presence of a suitably oriented lysine leads to the condensation of the -amine with the hapten. The formation of enaminone at the active site results in a molecular imprint that leads to the production of antibodies that function as aldol catalysts via a lysine-dependent class I aldolase mechanism (Eq. 8B2.12). 

Aldolases such as fructose-1,6-bisphosphate aldolase (FBP-aldolase), a crucial enzyme in glycolysis, catalyze the formation of carbon-carbon bonds, a critical process for the synthesis of complex biological molecules. FBP-aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate (DHAP) and glyceralde-hyde-3-phosphate (G3P) to form fructose-1,6-bisphosphate. There are two classes of aldolases the first, such as the mammalian FBP-aldolase, uses an active-site lysine to form a Schiff base, whereas the second class features an active-site zinc ion to perform the same reaction. Acetoacetate decarboxylase, an example of the second class, catalyzes the decarboxylation of /3-keto acids. A lysine residue is required for good activity of the enzyme the -amine of lysine activates the substrate carbonyl group by forming a Schiff base. 

In proteins with a symmetric structure, circular permutation can account for the shift of active-site residues over the course of evolution. A very good model of symmetric proteins are the (/Ja)8-barrel enzymes with their typical eightfold symmetry. Circular permutation is characterized by fusion of the N and C termini in a protein ancestor followed by cleavage of the backbone at an equivalent locus around the circular structure. Both fructose-bisphosphate aldolase class I and transaldolase belong to the aldolase superfamily of (a/J)8-symmetric barrel proteins both feature a catalytic lysine residue required to form the Schiff base intermediate with the substrate in the first step of the reaction (Chapter 9, Section 9.6.2). In most family members, the catalytic lysine residue is located on strand 6 of the barrel, but in transaldolase it is not only located on strand 4 but optimal sequence and structure alignment with aldolase class I necessitates rotation of the structure and thus circular permutation of the jS-barrel strands (Jia, 1996). 

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