Fructose- 1,6-bisphosphate aldolase

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]. 

Literally hundreds of aldehydes have so far been tested successfully by enzymatic assay and preparative experiments as a replacement for (18) in rabbit muscle FruA catalyzed aldol additions [16,25], and most of the corresponding aldol products have been isolated and characterized. The rabbit FruA can discriminate racemic dl-(18), its natural substrate, with high preference for the D-antipode, but kinetic enantioselec-tivity for nonionic aldehydes is rather low [84,89]. 

Functionally related to FruA is the novel class I fructose 6-phosphate aldolase (FSA) from E. coli, which catalyzes the reversible cleavage of fructose 6-phosphate (30) to give dihydroxyacetone (31) and d-(18) [90]. It is the only known enzyme that does not require the expensive phosphorylated nucleophile DHAP for synthetic purpose. 

---

The structure of human muscle fructose-1,6-bisphosphate aldolase, as determined by X-ray crystallography and downloaded from the Protein Data Bank. (PDB ID 1ALD Gamblin, S. J., Davies, G. J., Grimes, J. M., Jackson, R. M., Littlechild, J. A., Watson, H. C. Activity and specificity of human aldolases. J. Mol. Biol. v219, pp. 573-576, 1991.)... 

Tabic 2. Fructose 1,6-Bisphosphate Aldolase Catalyzed Additions of Dihy-droxyacelone Phosphate to Sugar Phosphates... 

Figure 10.14 Natural glycolytic substrates of the fructose 1,6-bisphosphate aldolase (FruA) and fructose 6-phosphate aldolase (FSA).

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]. 

This reaction is followed by another phosphorylation with ATP catalyzed by the enzyme phosphofructoki-nase (phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction may be considered to be functionally irreversible under physiologic conditions it is both inducible and subject to allosteric regulation and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are inter-converted by the enzyme phosphotriose isomerase. 

Antonijczuk, K., O.S. Kroftova, A.H. Varghese, A. Antonijczuk, D.C. Henjum, G. Korza, J. Ozols, and W.F. Sunderman, Jr. 1995. The 40kDa 63Ni2+-binding protein (pNiXc) on Western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A. Biochim. Biophys. Acta 1247 81-89. 

Transaldolase, which catalyzes reactions with d-erythrose 4-phosphate and D-fructose 6-phosphate as substrates. As in the case of fructose-1,6-bisphosphate aldolase, this enzyme uses a e-amino side-chain to form a Schiff base intermediate. In this case, however, the triose phosphate moiety is not released but is transferred to the other aldose (in this case, the aldotetrose). 

Fructose-1,6-bisphosphate aldolase Class 1 Aldolases Imine... 

SORBITOL DEHYDROGENASE FRUCTOSE-1,6-BISPHOSPHATASE FRUCTOSE-2,6-BISPHOSPHATASE D-Fructose 2,6-bisphosphate, 6-PHOSPHOFRUCTO-2-KINASE Fructose-1,6-bisphosphate aldolase, ALDOLASE... 

Lemaire et al. have developed a efficient fructose-1,6-bisphosphate aldolase (FBPA)-mediated synthesis of aminocyclitol analogs of valiolamine [34], This one-pot route involves the formation of two C—C bonds where four stereocenters are created. The first C—C bond formation reaction is catalyzed by the aldolase, coupling DHAP to nitrobutyraldehydes the other one is the result of a highly stereoselective intramolecular Henry reaction occurring on the intermediate nitroketone under acidic conditions during the aldolase-catalyzed reaction and phytase-catalyzed phosphate hydrolysis coupled step (Scheme 4.13). 

Starting from commercially available fructose 1,6-bisphosphate and hydroxypyruvate, three enzymes were in use fructose 1,6-bisphosphate aldolase, triosephosphate isomerase, and transketolase see E. T. Zimmermann,... 

Table 4.6.6 Control values for fructose-1-phosphate and fructose-1,6-bisphosphate aldolases [36]...

Cleavage of Fructose 1,6-Bisphosphate The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensation (p. 485). Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde 3-phosphate, an aldose, and dihydroxyacetone phosphate, a lcetose ... 

Closely related to aldolases is transaldolase, an important enzyme in the pentose phosphate pathways of sugar metabolism and in photosynthesis. The mechanism of the transaldolase reaction (Eq. 17-15) is similar to that used by fructose-1,6-bisphosphate aldolase with a lysine side chain forming a Schiff base and catalytic aspartate and glutamate side chains.198... 

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

Fig. 8. Stability of the rhamnulose 1-phosphate aldolase from Escherichia coli (RhuA) vs. that of the fructose 1,6-bisphosphate aldolase from rabbit muscle (FruA) in phosphate buffer (pH 7.2 25°C ca. 1 Uml ) a) RhuA b) O RhuA, 30% EtOH c) RhuA, 50% DMSO d) FruA...

In vivo, the D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes the pivotal reaction of the glycolysis pathway the equilibrium addition of 41 to D-glyceraldehyde 3-phosphate (GA3P, 12) to give D-fructose 1,6-bisphosphate (42) [43]. The equilibrium constant of 104 M 1 strongly favors synthesis [229]. 

Table 4. Substrate tolerance of fructose 1,6-bisphosphate aldolase...

Willnow P (1984) Fructose-1,6-bisphosphate Aldolase. In Bergmeyer H-J (ed) Methods of Enzymatic Analysis. 3rd edn. Verlag Chemie, Weinheim, vol. 4, p 346... 

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. 

---