Class I aldolases

A major group of C-C-bonding enzymes consists of aldolases which serve in various metabolic pathways, mainly in the direction of aldol cleavage. Aldol con- 

Residues Glu224 and Asp263 are conserved among phosphonopyruvate and sulfopyruvate decarboxylases and are shaded in green. 

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Aldolases cataly2e the asymmetric condensation of intermediates common in sugar metaboHsm, such as phosphoenolpymvic acid, with suitable aldehyde acceptors. Numerous aldolases derived from plants or animals (Class I aldolases) or from bacteria (Class II) have been examined for appHcations (81). Efforts to extend the appHcations of these en2ymes to the synthesis of unusual sugars have been described (2,81). 

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. 

The Chemical Evidence for the Schiff Base Intermediate in Class I Aldolases... 

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. 

Figure 10.3 Schematic mechanism of class I aldolases (a) and of class II aldolases (b).

Schurmann, R. and Sprenger, G.A. (2001) Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases. The Journal of Biological Chemistry, 276, 11055-11061. 

Although fructose-6-phosphate aldolase (FSA) does not belong to the DHAP-dependent aldolases group, it deserves to be mentioned in this chapter as it can be considered as an alternative to those enzymes, or at least, an alternative to FBPA. FSA was described for the first time by Schiirmann and Sprenger in E. coli K-12 strain MG1655 [50]. The enzyme is a class I aldolase with a homodecameric... 

There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2 the Zn2+ polarizes the carbonyl group... 

MECHANISM FIGURE 14-5 The class I aldolase reaction. The reaction shown here is the reverse of an aldol condensation. Note that cleavage between C-3 and C-4 depends on the presence of the carbonyl group at C-2. and (2)The carbonyl reacts with an active-site Lys residue to form an imine, which stabilizes the carbanion generated by the bond cleavage—an imine delocalizes electrons even better than... 

Aminocatalysis is a biomimetic strategy used by enzymes such as class I aldolases. Application of aminocatalysis in an asymmetric aldol reaction was reported in the early 1970s. Proline (19) efficiently promoted an intramolecular direct aldol reaction to afford Wieland-Miescher ketone in 93% ee [17,18]. More than 25 years later, in 2000, List, Barbas, and co-workers reported that proline (19) is also effective for intermolecular direct aldol reactions of acetone (le) and various aldehydes 3. Notably, the reaction proceeded smoothly in anhydrous DMSO at an ambient temperature to afford aldol adducts in good yield and in modest to excellent enantioselectivity (up to >99% ee, Scheme 9) [19-22]. The chemical yields and selectivity of proline catalysis are comparable to the best metallic catalysts, although high catalyst loading (30 mol %) is required. Proline (19)... 

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). 

Very recently the group of Resmini described the development of imprinted microgels mimicking a class I aldolase in the catalysis of the aldol condensation between 4-nitrobenzaldehyde and acetone [79]. They prepared highly crosslinked microgels (80 mol%) based on /V,/V -methyIenebisaery 1 amide as the crosslinker and containing as catalytically active monomer a proline derivative (Fig. 5). 

G. Schneider, Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the a/ P-barrel within the class I aldolase family, Structure 1996, 4, 715-724. 

The formation of covalent substrate-catalyst adducts might occur, e.g., by single-step Lewis-acid-Lewis-base interaction or by multi-step reactions such as the formation of enamines from aldehydes and secondary amines. The catalysis of aldol reactions by formation of the donor enamine is a striking example of common mechanisms in enzymatic catalysis and organocatalysis - in class-I aldolases lysine provides the catalytically active amine group whereas typical organocatalysts for this purpose are secondary amines, the most simple being proline (Scheme 2.2). 

Although the transition state analog approach is suitable for enzymes that bind their transition state noncovalendy, many natural enzymes achieve rate accelerations through covalent catalysis. For example, in the mechanism of most esterases and amidases, a functional group (e.g., a serine hydroxyl) of the protein covalently interacts with the substrate to form a protein bound intermediate. Furthermore, nature s most fundamental carbon-carbon bond-forming enzymes, class I aldolases, use... 

In our original hapten design for aldolase antibodies, the /3-diketone functionality of hapten 4 was used as a reactive immunogen to trap a chemically reactive lysine residue in the active site of an antibody as a stable enaminone. The chemical mechanism leading up to the stabilized enaminone should match that of Class I aldolases over this portion of the reaction coordinate. 

We have also examined the use of cyclodextrin-derived artificial enzymes in promoting bimolecular aldol reactions, specifically those of m-nitrobenzaldehyde (57) and ofp-t-butylbenzaldehyde (58) with acetone [141]. Here, we examined a group of mono-substituted cyclodextrins as catalysts (e.g. 59), as well as two disubstituted (3-cyclodextrins (e.g. 60) (10 catalysts in all). They all bound the aldehyde components in the cyclodextrin cavity and used amino groups of the substituents to convert the acetone into its enamine. An intracomplex reaction with 58 and hydrolysis of the enamine product then afforded hydroxyketone 61 (cf. 62). These catalysts imitate natural enzymes classified as Class I aldolases. 

Mechanistically, the antibody aldolases resemble natural class I aldolase enzymes (Scheme 4.7) [52]. In the first step of a condensation reaction, the s-amino group of the catalytic lysine reacts with a ketone to form a Schiffbase. Deprotonation of this species yields a nucleophilic enamine, which condenses with electrophilic aldehydes in a second step to form a new carbon-carbon bond. Subsequent hydrolysis of the Schiffbase releases product and regenerates the active catalyst. 

The reaction of the glycolytic enzyme fructose-1,6-bis-phosphate aldolase, perhaps the best-known class I aldolase, is believed to proceed according to the mechanism shown in Scheme 12131. 

Initial evidence for the structural diversification of the /fa-barrel fold by circular permutation was found in sequence alignments of members of the a-amylase superfamily [31]. More recently, analysis of crystallographic data of transaldolase B from E. coli suggested that the enzyme was derived from circular permutation of a class I aldolase [32]. In either case, the shift of the two N-terminal /fa-repeats (plus the /(-strand of the third subunit for amylases) onto the C-terminus resulted in no apparent functional changes. 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

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