Aldolase substrate specificity

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. 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

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). 

The hexose phosphate, fructose-1,6-diphosphate, is split by aldolase into two triose phosphates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Aldolase consists of four 40-kDa subunits. Three tissue-specific forms exist in human tissues aldolase A (ubiquitous and very active in the muscle), aldolase B (liver, kidney, and small intestine), and aldolase C (specific to the brain). These three isozymes have nearly the same molecular size but differ in substrate specificity,... 

DeSantis, G., Liu, J., Clark, D.R et al. (2003) Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase. Bioorganic and Medicinal Chemistry, 11, 43-52. 

Figure 6.1 Alteration of substrate specificity of sialic acid aldolase by directed evolution...

The work described in this chapter illustrates that several approaches can successfully achieve the goal of broadening the substrate scope of aldolases. Whereas these enzymes have been perceived as being useful only in very specific applications due to their strict substrate specificity, it is becoming clear that they can in fact be versatile, practical biocatalysts that can be applied to a wider range of synthetic problems. 

The key enzymes involved in these conversions are transaldolase and transketolase. The two enzymes are similar in their substrate specificities. Both require a ketose as a donor and an aldose as an acceptor. The steric requirements at positions C-1 through C-4 are the same as the requirements of aldolase in the glycolytic pathway, except that aldolase requires phosphorylation at C-1, and both transaldolase and transketolase require a free hydroxyl group at C-1. 

To expand the substrate specificity and stereoselectivity of the aldolase DERA (2-deoxyribose-5-phosphate aldolase, E.C. 4.1.2.4), both site-specific mutagenesis and random mutagenesis have been investigated (DeSantis, 2003). The goal was to extend substrate specificity to the unnatural non-phosphorylated substrate, D-2-deoxyribose. 

The new aldolase differs from all other existing ones with respect to the location of its active site in relation to its secondary structure and still displays enantiofacial discrimination during aldol addition. Modification of substrate specificity is achieved by altering the position of the active site lysine from one /3-strand to a neighboring strand rather than by modification of the substrate recognition site. Determination of the 3D crystal structure of the wild type and the double mutant demonstrated how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate. It is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. 

Aldolase antibodies obtained by reactive immunization are notable for high activity, broad substrate specificity, and high selectivities [53]. Rate accelerations are typically in the range 105 to 107-fold over background. Although the k /K values are 102 to 104 lower than those of aldolase enzymes, these are among the most efficient antibody catalysts described to date. Their efficacy is all the more notable in light of the inherently complex, multistep process they catalyze. 

Despite the broader substrate tolerance for aldehyde acceptors by DHAP-dependent aldolases, the donor substrate specificity is narrow. With the exception of DHAP, only a few donors have shown to be acceptable as weak substrates (Scheme 5.6).20a 21a 26... 

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. 

Lrthreonine aldolase (L-threonine acetaldehyde-lyase) catalyzes the reversible condensation of acetaldehyde and glycine to form L-threonine. The enzyme has been shown to be an activity distinct from serine hydroxy-methyltransferase that also catalyzes the above reaction (85,86). The substrate specifically of the adolase has been demonstrated to be flexible with respect to the aldehyde involved. The enzyme has been shown to form phenylserine derivatives from substituted benzaldehydes and glycine (86). 

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