Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.     

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.     

Mechanism of the Class I KDPG aldolase

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.     

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.     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

Classification of fructose-1,6-bisphosphate aldolases based on 18O retention in the cleavage reaction.

Oxygen (18) was used as a mechanistic probe in the investigation of several different sources of fructose 1,6-bisphosphate aldolases (EC 4.1.2.13) which, due to differences in some physical and chemical properties, could not be clearly put in either Class I or Class II. Aldolases may be identified as belonging to a particular class on the basis of the amount of 180 retained in the dihydroxyacetone phosphate produced in the cleavage of [2-Oxygen (18)] fructose 1,6-biphosphate. The mechanism of Class I aldolases involves an obligatory exchange of the C-2 oxygen atom of fructose 1,6-bisphosphate, leading to the absence of 180 in the product. For Class II aldolases, the C-2 oxygen atom is retained in the aldol cleavage reaction. Aldolases from spinach and L. casei base intermediate. Aldosase from C. perfringens was found to be Class II, suggesting a metal-chelate intermediate. Results with Euglena aldolase confirmed that this organism contained both types of aldolases with approximately 78% Class II. The data show that despite a wide variety of physical and chemical properties, there are important mechanistic similarities within each class of enzyme and significant differences between the two classes. The determination of 180 retention in the product of the cleavage reaction using [2-180] fructose 1,6-biphosphate is an accurate means of classifying these enzymes since it is a measure of a property which is directly related to the mechanisms of the reactions.     

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.     

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.     

An unusual class I (Schiff base) fructose-1,6-bisphosphate aldolase from the halophilic archaebacterium Haloarcula vallismortis

An electrophoretically homogeneous class I (Schiff base) alsolase has been isolated for the first time from the archaebacterial halophile Haloarcula (Halobacterium) vallismortis. The aldolase was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity and kinetic properties. The subunit mass of aldolase is 27 kDa, which is much smaller than other class I aldolases. By the gel filtration method, the molecular mass of the halobacterial enzyme was estimated as 280 +/- 10 kDa, suggesting a decameric nature. In contrast to many halobacterial proteins, the H. vallismortis aldolase, though a halophilic enzyme, did not show an excess of acidic residues. Unlike the eukaryotic aldolases, the activity of the halobacterial enzyme was not affected by carboxypeptidase digestion. The general catalytic features of the enzyme were similar to its counterparts from other sources. No antigenic similarity could be detected between the H. vallismortis aldolase and class I aldolase from eubacteria and eukaryotes or class II halobacterial aldolases.     

A comparative study of aldolase from human muscle and liver

Aldolase was purified from human skeletal muscle and human liver by techniques capable of processing large quantities (10-20kg) of tissue. The methods used also proved convenient for isolating aldolase on a large scale from other mammalian and avian sources. Aldolase from both human liver and muscle was crystallized; each gave two crystalline forms, depending on the conditions of crystallization. X-ray studies on the muscle aldolase crystals suggest a close structural similarity between human and rabbit muscle aldolase. Aldolases from human muscle and liver have similar pH optima and pH stability but their stability to heat treatment differs. The effect of heat on the enzymes may therefore provide an easy means of distinguishing them. The kinetic constants K(m) and k(cat.) for these aldolases are similar to other mammalian aldolases. Amino acid analyses and tryptic peptide ;mapping' show that the primary structures of the two aldolases differ greatly.     

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.     

Crystal structures of LacD from Staphylococcus aureus and LacD.1 from Streptococcus pyogenes: insights into substrate specificity and virulence gene regulation

Staphylococcus aureus LacD, a Class I tagatose-1,6-bisphosphate (TBP) aldolase, shows broadened substrate specificity by catalyzing the cleavage of 1,6-bisphosphate derivatives of d-tagatose, d-fructose, d-sorbose, and d-psicose. LacD.1 and LacD.2 are two closely-related Class I TBP aldolases in Streptococcus pyogenes. Here we have determined the crystal structures of S. aureus LacD and S. pyogenes LacD.1. Monomers of both enzymes are folded into a (β/α)₈ barrel and two monomers associate tightly to form a dimer in the crystals. The structures suggest that the residues E189 and S300 of rabbit muscle Class I fructose-1,6-bisphosphate (FBP) aldolase are important for substrate specificity. When we mutated the corresponding residues of S. aureus LacD, the mutants (L165E, L275S, and L165E/L275S) showed enhanced substrate specificity toward FBP.

Structured summary

lacDbinds to lacD by X-ray crystallography(View interaction)lacD1binds to lacD1 by X-ray crystallography(View interaction)     

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.     

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.     

Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.     

Properties of Aldolase from Francisella tularensis

The aldolase of Francisella tularensis resembles Class II aldolases in its requirement for divalent ions and its inactivation by metal chelating agents. Cysteine and other reducing agents stimulated the activity of the enzyme.