Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

Abstract

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

“…The difference in the number of methionine residues between the predicted and the experimentally determined amino acid composition is most likely due to a proteolytic processing at the amino terminus [25]. There is a remarkable similarity in the overall amino acid composition to the E. coli aldolase 2 [26], which may be reflected also in a sequence homology and might be the reason for the existence of immuno-reactive material of about 35 kDa in E. coli extracts probed with anti-aldolase antiserum (Fig. 1).…”

“…In order to answer the question whether or not isoenzymes of aldolase, as found in higher organisms [l] and also in E. coli [26], do exist in yeast, a gene disruption was performed by inserting the yeast URA3 gene into the HpaI site of one chromosomal copy of the FBAl gene in the homozygous diploid ura3 strain W303D. Integration of the URA3 gene into the FBAI locus in the resulting strain HGlOl was monitored by Southern and Northern blotting (Fig.…”

Cloning, sequencing, and characterization of the gene encoding the class I fructose-1,6-bisphosphate aldolase of Staphylococcus carnosus

“…This complex can be fixed by reduction with NaBH4, leading to an irreversible inactivation of the enzyme (16 (15); and they can be inhibited by EDTA (16). Since it was believed that class I aldolases were restricted to higher eucaryotic organisms, it was a surprise to find them in Peptococcus aerogenes (20), Lactobacillus casei (21), Escherichia coli (33), and most staphylococcal species (12).…”

“…This sequence is highly conserved not only in the S. carnosus but also in other FBP aldolases (Fig. 3) (30,33), was used for complementation experiments. E. coli NP315 (Fda-) was grown in M9 medium (27) supplemented with 0.4% glucose, 0.01 M MgSO4, and 0.2% (NH4)2SO4 at 30°C.…”

Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae)

“…Class II aldolases occur in fungi and procaryotes (6,23). In some procaryotes, such as Escherichia coli, an additional class I type aldolase can be developed upon growth on pyruvate or lactate (26). On the other hand, algae appear to have both a class I and a class II aldolase (2,5,8,15,18,(21)(22)(23).…”