Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

Abstract

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.

The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase

“…For example, the mutant subunit could have a higher affinity for wild-type than mutant enzyme, leading to many more mixed dimers than expected on statistical grounds, or may be able to associate with other aldolase subunits following partial denaturation, thus permitting it to catalyze the denaturation of many of the remaining wild-type subunits. Alternatively, Fda might act as a tetramer in vivo, although it has been reported to sediment as a dimer in vitro (5,6,31). The low amount of wild-type aldolase activity that we observed in the mutant/wild-type merodiploid would be expected if Fda were a tetramer and a single mutant Fda monomer was able to poison the tetramer.…”

From prebiotic chemistry to cellular metabolism—The chemical evolution of metabolism before Darwinian natural selection

“…It is interesting to see that in some cases, the same stoichiometric mechanism can be performed by two different catalytic mechanisms. This occurs, for instance, in type I aldolases, which are catalyzed by a lysine residue of the enzyme, and type II aldolases which are catalyzed by zinc (Rutter, 1964;Baldwin et al, 1978) and in hydrolysis reactions, where the same stoichiometric mechanism (a nucleophylic attack of HO À ) is achieved by a serine residue in serine proteinases (see Wharton, 1998), or by H 2 O in aspartic proteinases (see Meek, 1998). This feature is stated in Hypothesis 1.…”

A Class II fructose-1 ,6-bisphosphate aldolase from a halophilic archaebacterium Haloferax mediterranei.

“…Co 2ϩ could restore only 20% activity. In most Class II aldolases, viz., Aspergillus (Jaganathan, 1956), S. cerevisiae , B. stearothermophilus (Hill et al, 1976), E. coli (Baldwin et al, 1978), and E. gracilis (cytosol enzyme) (PelzerReith et al, 1994) , as in S. cerevisiae , Aspergillus niger (Jagannathan, 1956), B. stearothermophillus (Hill et al, 1976), and E. coli (Berry and Marshall, 1993). In some other aldolases, viz., Clostridium perfringens and Vibrio marinus (Jones et al, 1979), the divalent metal is Co 2ϩ , and in cyanobacteria (Willard and Gibbs, 1975) and Cl.…”

“…The subunit molecular mass of most Class II aldolases is 40 kDa, as in yeast (Harris et al, 1969), E. coli (Baldwin et al, 1978), and E. gracilis (PelzerReith et al, 1994). However, in some bacilli, e.g., B. stearothermophilus (Sugimoto and Nosoh, 1971) and Bacillus subtilis (Ujita, 1978), the subunit mass is 30 kDa.…”

“…Subsequently, with the cloning of the fda gene of E. coli, structural and sequencing studies have added to the information of Class II aldolases (Alefounder et al, 1989;Berry and Marshall, 1993;Naismith et al, 1992). The Class II aldolases that have been purified to date, either from a eubacterium, e.g., E. coli (Baldwin et al, 1978) and Bacillus stearothermophilus (Hill et al, 1976), or from a eukaryote, viz., S. cerevisiae (Harris et al, 1969), show a similarity in their requirement for a divalent metal ion for activity and in their inhibition by EDTA and other metal chelators.…”