Ketohexokinase (ATP:D-fructose 1-phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of [ gamma-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of the enzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detected between the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.     

Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria

Two fructose-1,6-bisphosphate aldolases from the acido- and thermophilic red alga Galdieria sulphuraria were purified to apparent homogeneity and N-terminally microsequenced. Both aldolases had similar biochemical properties such as Km (FBP) (5.6-5.8 microM) and molecular masses of the native enzymes (165kDa) as determined by size exclusion chromatography. The subunit size of the purified aldolases, as determined by SDS-PAGE, was 42kDa for both aldolases. The isoenzymes were not inhibited by EDTA or affected by cysteine or potassium ions, implying that they belong to the class I group of aldolases, while other red algae are known to have one class I and one class II aldolase inhibited by EDTA. cDNA clones of the cytosolic and plastidic aldolases were isolated and sequenced. The gene for the cytosolic isoenzyme contained a 303bp untranslated leader sequence, while the gene for the plastidic isoenzyme exhibited a transit sequence of 56 amino-acid residues. Both isoenzymes showed about 48% homology in the deduced amino-acid sequences. A gene tree relates both aldolases to the basis of early eukaryotic class I aldolases. The phylogenetic relationship to other aldolases, particularly to cyanobacterial class II aldolases, is discussed.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

TWO CLASS I ALDOLASES IN KLEBSORMIDIUM FLACCIDUM (CHAROPHYCEAE): AN EVOLUTIONARY LINK FROM CHLOROPHYTES TO HIGHER PLANTS1

Two fructose-bisphosphate aldolases(EC 4.1.2.13) from Klebsormidium flaccidum Silver, Mattox and Black-well were purified by affinity elution from phosphocellulose. The two enzymes were subsequently separated by HPLC on an anion-exchange column (QAE-silica). The aldolase eluting first represented 5% of the total activity; the other aldolase represented the remaining activity. The activity of the enzymes was not reduced by the presence of 1 mM EDTA or increased by 0.1 mM Zn²⁺, establishing their character as class I type (Me²⁺ independent) aldolases. The K_m(fructose-1,6-bisphosphate) values were 1.7 and 34.7 μM for the enzyme eluting first and second, respectively, from the QAE-silica column. The subunit molecular masses, as determined by SDS-PACE, were 40.5 and 37 kD; the specific activities of the purified enzymes were 7.9 and 24.7 · mg^?1 protein, respectively. The two aldolases of K. flaccidum are homologous to the cytosol and chloroplast specific isoenzymes of higher plants by several criteria and are therefore probably located in the same cellular compartments in K. flaccidum. The K_m and specific activity for the chloroplast aldolase of K. flaccidum are three times higher than for the chloroplast aldolase of higher plants, a remarkable difference. Immunotitration with specific antisera against the chloroplast aldolase of Chlamydomonas reinhardtii Dangeard and spinach showed that the chloroplast aldolase of K. flaccidum was immunochemically intermediate in structure to the respective aldolases of C. reinhardtii and higher plants. K. flaccidum is the second species of Charophyceae (besides Chara foetida Braun) with two class I aldolases as in higher plants whereas two species of Chlorophyceae have only one class I aldolase and, under some conditions, an additional class II (Me²⁺ dependent) aldolase. Thus, aldolases may turn out, in addition to the known enzymes of glycolate conversion and urea degradation, be a novel enzyme system to evaluate algal evolution along with cytological features.     

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.     

Structural Similarities between Spinach Chloroplast and Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases : Immunochemical and Amino-Terminal Amino Acid Sequence Analysis

Immunochemical studies using polyclonal antisera prepared individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases showed significant cross reaction between both forms of spinach aldolase and their heterologous antisera. The individual cross reactions were estimated to be approximately 50% in both cases under conditions of antibody saturation using a highly sensitive enzyme-linked immunosorbent assay. In contrast, the class I procaryotic aldolase from Mycobacterium smegmatis and the class II aldolase from yeast (Saccharomyces cerevisiae) did not cross-react with either type of antiserum. The 29 residue long amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined. Comparisons of these sequences with those of other aldolases showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the corresponding region of the M. smegmatis enzyme, especially in that region which forms the first “beta sheet” in the secondary structure of the eucaryotic aldolases. Moreover, results of a systematic comparison of the amino acid compositions of a number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase belongs to the eucaryotic rather than the procaryotic “family” of class I aldolases.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Purification and properties.

Fructose diphosphate aldolase has been purified to homogeneity from Mycobacterium smegmatis. Physicochemical studies showed that the enzyme is a tetramer of molecular weight 158,000. Mycobacterium smegmatis aldolase, though a bacterial enzyme, possesses properties similar to other class I aldolases. Inactivation of the enzyme by sodium borohydride in presence of dihydroxyacetone phosphate suggested the formation of a Schiff-base intermediate.     

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

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.     

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

Aldolase activity of Chara foetida (Braun) could be separated into a minor (peak I) and a major peak (peak II) by ion-exchange chromatography on DEAE-cellulose. Affinity chromatography on P-cellulose resulted in highly purified aldolase preparations with specific activities of 3.2 and 4.8 units per milligram protein and molecular subunit masses of 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak I and peak II, respectively. Both aldolases belong to class I aldolase since the activity is not inhibited by 1 millimolar EDTA. The K_m (fructose-1,6-bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The aldolase of peak I showed a 6.7 times stronger crossreaction with a specific antiserum against the cytosol aldolase of spinach than with an antiserum against the chloroplast aldolase of spinach. On the other hand the aldolase of peak II showed a 5.1 times stronger cross-reaction with the α-plastidaldolase antiserum than with the α-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me²⁺ independent) and class II (Me²⁺ dependent) aldolase in other algae.     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.     

Characterization of a Mn-dependent Fructose-1,6-bisphosphate Aldolase in Deinococcus radiodurans

The key enzyme of the glycolytic pathway of Deinococcus radiodurans, fructose-1,6-bisphosphate aldolase, could be induced independently by glucose and Mn. The enzyme exhibited the characteristics of the metal-dependent Class II aldolases. Unlike most Class II aldolases, the deinococcal aldolase preferred Mn, not Zn, as a cofactor. The fbaA gene encoding the deinococcal aldolase was cloned and the protein overproduced in various Escherichia coli expression hosts. However, the overexpressed deinococcal enzyme aggregated and formed inclusion bodies. Dissolving these inclusion bodies by urea and subsequent purification by nickel affinity chromatography, resulted in a protein fraction that exhibited aldolase activity only in the presence of Mn. This active aldolase fraction exhibited masses of about 70 kDa and 35 kDa by gel filtration and by SDS gel electrophoresis, respectively, suggesting that the active aldolase was a dimer.     

Studies on aldolase from human cardiac tissue

Fructose diphosphate aldolase has been purified to homogeneity from human cardiac tissue. Physicochemical studies show that the enzyme is a tetramer of molecular weight 160 000 and possesses properties common to other Class I aldolases. Catalytic studies, together with amino acid analysis and tryptic peptide fingerprints, suggest that the enzyme is a typical Type A, muscle aldolase. Contrary to earlier reports, no other form of aldolase could be identified in adult human heart.     

Archaebacterial class I and class II aldolases from extreme halophiles

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity fromHalobacterium halobium, H. salinarium, H. cutirubrum, H. mediterranei andH. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase fromH. saccharovorum, Halobacterium R-113, H. vallismortis andHalobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.     

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP synthase

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F1 moiety from the Escherichia coli ATP synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20-22 mol/100 mol). Peptide mapping of subunits I and II denatured with sodium dodecyl sulfate showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F1 ATPase from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, halobacteria in general, possess an ATPase which is unlike the ubiquitous F0F1 ATP synthase.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Purification and characterization of a class I fructose 1,6-bisphosphate aldolase from Staphylococcus carnosus

Summary A fructose 1,6-bisphosphate aldolase (E.C.4.1.2.13) from Staphylococcus carnosus DSM 20501 was purified for the first time. The enzymatic activity was insensitive to high levels of EDTA indicating that the enzyme is a class I aldolase. This enzyme exhibits good stability at high temperatures and extreme stability over a wide pH range. The K _m for fructose 1,6-bisphosphate as substrate was 0.022 mm. The S. carnosus aldolase is a monomeric enzyme with a molecular mass of about 33 kDa. It exhibits a relatively broad pH optimum between pH 6.5 and 9.0. Furthermore, the aldolase accepts other aldehydes in place of its natural substrate, glyceraldehyde 3-phosphate, allowing the synthesis of various sugar phosphates. Offprint requests to: M. R. Kula     

Distribution of class I and class II fructose biphosphate aldolases in halophilic archaebacteria

Abstract Both class I (Schiff base-forming) and class II (metal-requiring) fructose biphosphate (FDP) aldolases were found to be distributed among halophilic archaebacteria. The type of enzyme did not vary with the growth medium. The aldolase activities were also halophilic.     

Properties of fructose 1,6-diphosphate aldolase from Drosophila melanogaster.

The amino acid composition and other properties of fructose 1,6-diphosphate aldolase from pupae of Drosophila melanogaster are reported and compared with those of other class I aldolases. Drosophila aldolase subunits contain only four residues of cysteine, five histidines, and two methionines. All four cysteine side chains react with 5,5′-dithiobis(2-nitrobenzoic acid) only in the presence of denaturating agent and are therefore thought to be buried within the molecule. With bromoacetate one carboxymethyl group is incorporated in the native enzyme with the loss of 90% of catalytic activity; inorganic phosphate is partially inhibiting this reaction. The near-uv absorption spectra of Drosophila and rabbit muscle aldolases are similar, the insect enzyme having higher absorbancies over the entire region corresponding to its higher tryptophan content. Circular dichroism-spectra of Drosophila aldolase indicate an α-helix content of 26%. Both the insect and vertebrate enzymes display marked tryptophan ellipticity bands between 290 and 300 nm.