Class II aldolases

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 

Certainly, a detailed understanding of the contributions of active-site residues in substrate recognition and the catalytic event was highly desirable. 

I in a process similar to the photorespiration that is caused by the oxygen-consuming side reaction of D-ribulose 1,5-bisphosphate carboxylase [13], and therefore cannot be used for syntheses. 

In concurrent efforts in collaboration with G.E. Schulz s group, the two Class 

II aldolases FucA and RhuA from E. coli have been crystallized solution of their spatial structures confirmed a close similarity in their overall fold [14]. Both enzymes are homotetramers in which subunits are arranged in C4 symmetry. The active site is assembled in deep clefts at the interface between adjacent subunits, and the catalytic zinc ion is tightly coordinated by three His residues. From X-ray 

---

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. 

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. 

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

The D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes in vivo the equilibrium addition of (25) to D-glyceraldehyde 3-phosphate (GA3P, (18)) to give D-fructose 1,6-bisphosphate (26) (Figure 10.14). The equilibrium constant for this reaction of 10 strongly favors synthesis [34]. The enzyme occurs ubiquitously and has been isolated from various prokaryotic and eukaryotic sources, both as class I and class II forms [30]. Typically, class I FruA enzymes are tetrameric, while the class II FruA are dimers. As a rule, the microbial class II aldolases are much more stable in solution (half-lives of several weeks to months) than their mammalian counterparts of class I (few days) [84-86]. 

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). 

Hernaez MJ, B Eloriano, JJ Rfos, E Santero (2002) Identification of a hydratase and a class II aldolase involved in biodegradation of the organic solvent tetralin. Appl Environ Microbiol 68 4841-4846. 

Kimura and co-workers have synthesized a series of alkoxide complexes with the alcohol functionality as a pendent arm.447 674 737 A zinc complex of l-(4-bromophenacyl)-l, 4,7,10-tetraaza-cyclododecane was also synthesized by the same workers to mimic the active site of class II aldolases. The X-ray structure shows a six-coordinate zinc center with five donors from the ligand and a water molecule bound. The ketone is bound with a Zn—O distance of 2.159(3) A (Figure 12). Potentiometric titration indicated formation of a mixture of the hydroxide and the enolate. Enolate formation was also independently carried out by reaction with sodium methoxide, allowing full characterization.738... 

Recoupling Cancer-Protective Properties of Angle-Spinning NMR High-Selenium Broccoli A Class II Aldolase MimH ... 

A class II aldolase-mimicking synthetic polymer was prepared by the molecular imprinting of a complex of cobalt (II) ion and either (lS,3S,4S)-3-benzoyl-l,7,7-trimethylbicyclo[2.2.1] heptan-2-one (4a) or (lR,3R,4R)-3-benzoyl-l,7,7-trimethylbicyclo[2.2.1]heptan-2-one (4b)... 

Scheme 4.1 Simplified representation of the reaction mechanisms of class I and class II aldolases.

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,... 

Scheme 2.2.5.2 Diastereoselective kinetic racemate resolution using the Class II aldolase FucA.

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/ ... 

The catabolism of L-fucose by E. coli requires cleavage of L-fuculose-l-phosphate to form dihydroxy-acetone phosphate and D-lactaldehyde by a class II aldolase.193... 

Fig. 16. Mechanistic model for DHAP addition to an aldehyde acceptor by class II aldolases based on the inhibitor-liganded FucA structure (right) in comparison to earlier proposals (left)...

Figure 9.11 Mechanism of action of the class II aldolase from E.cofi.

Figure 9.11 demonstrates the Schiffbase formation reaction for class II aldolases, which are mainly found in prokaryotes E.coli aldolase uses a zinc ion to ionize the carbonyl group of DHAP (Alan Berry, University of Leeds) it is now Figure 9.12. 

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. 

Kimura and his associates have been preeminent in exploiting the potential of Zn(II) complexes of pendant-arm polyaza macrocycles to act as models for the hydrolytic Zn(II)-containing enzymes. Collectively, their work in this area involves structurally unmodified macrocycles as well as pendant-arm macrocycles, and the reader is referred to a number of reviews 6-15) that summarize their work in its entirety. The particular object of this section is to examine how different types of pendant arm have been introduced onto a macrocyclic framework and how it has been possible to utilize their presence to elicit information of relevance to a particular group of enzymes. The enzyme groups studied using pendant-arm macrocycles have been the alkaline phosphatases and the class II aldolases. 

Scheme 2 Proposed stabilizatioii of the enediolate form of dihydroxyacetone phosphate hy Zn(II) in the class II aldolase enz3une fructose l,6-bis(phosphate) aldolase, and its reaction with D-glyceraldehyde 3-phosphate to form n-fructose 1,6-bisphosphate.

Chen, L., Zhou, C., Yang, H., and Roberts, M.F., 2000, Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase. Biochemistry 39 12415-12423. 

As part of a biomimetic study for the enzyme class II aldolase" the equilibrium was reported for the cyclen complex, hydroxo-Zn(II)-l-(4-bromophenacyl)-l,4,7,10-tetraaza-cyclododecane and the intramolecular enolate 20, formed from Zn(II) and the enolate of l-(4-bromophenacyl)cyclen. This cyclization reaction was shown to be endothermic by 8.7 kJmol. However, with an entropy of 19 JmoU K it proceeds readily enough to show facile H/D exchange from the CH2 group of the exocyclic ligand. We wonder about the enthalpy and entropy changes associated with a different choice of other polyamines and/or central metals. 

---