Type II aldolases

Aldol reactions occur in many biological pathways, but are particularly important in carbohydrate metabolism, where enzymes called aldolases catalyze the addition of a ketone enolate ion to an aldehvde. Aldolases occur in all organisms and are of two types. Type 1 aldolases occur primarily in animals and higher plants type II aldolases occur primarily in fungi and bacteria. Both types catalyze the same kind of reaction, but type 1 aldolases operate place through an enamine, while type II aldolases require a metal ion (usually 7n2+) as Lewis acid and operate through an enolate ion. 

Figure 23.10 Mechanisms of type I and type II aldolase reactions in glucose biosynthesis.

Aldolases catalyze asymmetric aldol reactions via either Schiff base formation (type I aldolase) or activation by Zn2+ (type II aldolase) (Figure 1.16). The most common natural donors of aldoalses are dihydroxyacetone phosphate (DHAP), pyruvate/phosphoenolpyruvate (PEP), acetaldehyde and glycine (Figure 1.17) [71], When acetaldehyde is used as the donor, 2-deoxyribose-5-phosphate aldolases (DERAs) are able to catalyze a sequential aldol reaction to form 2,4-didexoyhexoses [72,73]. Aldolases have been used to synthesize a variety of carbohydrates and derivatives, such as azasugars, cyclitols and densely functionalized chiral linear or cyclic molecules [74,75]. 

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 Aldol reaction is one of the most powerful methods for creating the C-C bond. Typical conditions involve the formation of an enolate, usually with a stoichiometric equivalent of base. Stereoinduction is nsnally accomplished with chiral enolates, aldehydes, or auxiliaries.Nature, however, is much more efficient, having created enzymes that both catalyze the aldol reaction and produce stereospecific product. These enzymes, called aldolases, are of two types. The type II aldolases make use of a zinc enolate. Of interest for this section are the type I aldolases, which make use of enamine intermediates. Sketched in Scheme 6.6 is... 

FIGURE 10. (A) Dimeric structure of E. coli type II aldolase (PDB ID 1B57, Plate XXII). (B) The active site showing a bound transition-state inhibitor (phosphoglycolohydroxamate), Zn(II), the coordinated hgands and the catalyticaUy important Asp (Plate XXIII)... 

The enzymatic aldol reaction represents a useful method for the synthesis of various sugars and sugar-like structures. More than 20 different aldolases have been isolated (see Table 13.1 for examples) and several of these have been cloned and overexpressed. They catalyze the stereospecific aldol condensation of an aldehyde with a ketone donor. Two types of aldolases are known. Type I aldolases, found primarily in animals and higher plants, do not require any cofactor. The x-ray structure of rabbit muscle aldolase (RAMA) indicates that Lys-229 is responsible for Schiff-base formation with dihydroxyacetone phosphate (DHAP) (Scheme 13.7a). Type II aldolases, found primarily in micro-organisms, use Zn as a cofactor, which acts as a Lewis acid enhancing the electrophilicity of the ketone (Scheme 13.7b). In both cases, the aldolases accept a variety of natural (Table 13.1) and non-natural acceptor substrates (Scheme 13.8). 

SCHEME 13.7 (a) Type I aldolases form enamine nucleophiles (donor) (b) type II aldolases use Zn as a cofactor activating the aldehyde (acceptor). 

RAMA has been the most popular synthetic aldolase, due to its commercial availability. Notably, no significant differences in substrate specificity or stereoselectivity between FDP aldolases from different sources have been observed188. However, it is still important to verify this, especially for the type II aldolases which operate by a different mechanism. In fact, the type II aldolase from E. coli, which has been subcloned and overexpressed127, has the potential to supplant RAMA as the FDP aldolase of choice for synthesis. It has enhanced stability compared with RAMA (vide supra), and is available from a microbial as opposed to an animal source. Table 14.1-1 illustrates products prepared from FDP aldolase-catalyzed reactions with DHAP. 

Peptidase Models Phosphatase Models /J-Lactamase Models Type-II Aldolase Models More Recent Developments... 

Zn complexes " have been prepared that model type-II aldolases, which catalyze reversible aldol addition reactions. Enolizations are essential for the carbon-carbon bond formation in an aldol reaction, and Zn may play a role in such processes. For example, activation of the carbonyl of DHAP in the active site of type-II aldolases was performed by a Zn complex of l-(4-bromophenacyl)-cyclen (59) (Scheme 38). The pAa value for the acidic protons of the carbonyl a-position was 8.4. 

Fig. 6.53 Mechanism of type II aldolases illustrated for FucA and RhuA...

FDP Aldolase. The most extensively utilized class of enzymes for monosaccharide synthesis are the aldolases (E.C. sub-class 4.1.2.). This ubiquitous group of enzymes catalyzes reversible aldol reactions in vivo. Two major groups of aldolases exist type I aldolases, found primarily in higher plants and animals, catalyze aldol condensations by means of a Schiff base formed between an enzyme lysine e-amino group and the nucleophilic carbonyl group type II aldolases, found primarily in microorganisms, utilize a divalent zinc to activate the nucleophilic component (79). Approximately 25 aldolases have been identified to date (18),... 

Scheme 2.182 Mechanism of metal-dependent type II aldolases...

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