Novel kinetic and structural properties of the class-I D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

Investigation of aldolase 1, the class-I D-fructose 1,6-bisphosphate aldolase (EC4.1.2.13) from Escherichia coli (Crookes' strain), showed it to have unusual kinetic and structural properties. The enzyme appeared to be larger than was previously supposed and may be a decamer with a mol. wt. of approx. 340000. Its fructose 1,6-bisphosphate-cleavage activity was unaffected by these compounds. The enhancement exhibited a strong dependence on pH. These novel kinetic properties do not seem to be shared by any other fructose 1,6-bisphosphate aldolase, but recall the activation by polycarboxylic acids of the deoxyribose 3-phosphate aldolases from some other organisms. In view of its unusual properties, it is unlikely that aldolase 1 from E. coli is closely related to the class-1 aldolases that have been detected in several other prokaryotes, or to the typical class-1 enzymes from eukaryotes.     

Expression, purification, and characterization of natural mutants of human aldolase B. Role of quaternary structure in catalysis

Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.     

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.     

Molecular cloning, expression, purification, and characterization of fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis--a novel Class II A tetramer

The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.     

Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B

In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.     

Purification and properties of the native form of rabbit liver aldolase. Evidence for proteolytic modification after tissue extraction.

Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28 mumol/min per mg of protein and a fructose 1,6-bisphosphate cleavage of 20-28mumol/min per mg of protein and a frutose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42. This plasmid produces active rat aldolase C in the transfected E. coli host cells. The characteristics of the purified enzyme, e.g. mol. wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C. Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A. Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.     

Molecular cloning and expression of the Streptococcus lactis tagatose 1,6-bisphosphate aldolase gene in Escherichia coli

Abstract A 4.4 kb Eco RI DNA fragment of the Streptococcus lactis H₁ plasmid pDI1 was cloned into the Escherichia coli plasmid pACYC 184. The recombinant plasmid expressed d -tagatose 1,6-bisphosphate aldolase activity in E. coli . Enzyme activity was at the same level as in the original S. lactis host but was not repressed by glucose.     

Enzymatic properties of fish muscle aldolase

1. Aldolases were isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus, and carp Cyprinus carpio by ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and CM-Sepharose CL-6B columns, and examined for enzymatic properties. 2. The aldolases showed the highest activity in a pH range from 6.8-7.8 Km values for fructose-1,6-bisphosphate ranged from 0.025-0.10 mM. 3. Irrespective of fish species, aldolase activity was inhibited by ATP, ADP, and AMP. ATP showed the strongest inhibition and was competitive with fructose-1,6-bisphosphate. 4. The aldolases did not require divalent metal ions for activation and were completely inhibited at 0.1 mM Cu2+. 5. Thermal inactivation of the enzymes was of the first-order reaction. Red sea bream, Pacific mackerel and carp enzymes lost the activity by 50% when incubated at 50 degrees C for 8, 14 and 23 min, respectively.     

Carboxyl terminus region modulates catalytic activity of recombinant maize aldolase

Site-directed mutagenesis was utilized to study the functional role of the COOH-terminal region in recombinant maize aldolase. A single mutation was created in each of the last nine amino acids of the COOH terminus and characterized kinetically. Point mutations in the COOH-terminal region were found to influence both the rate of fructose 1,6-bisphosphate and fructose 1-phosphate cleavage. Catalytic efficiency, kcat/Km, was not affected by the mutations within experimental error consistent with this region of the COOH terminus modulating product release. Concentrations of the carbanion-enamine enzyme intermediate complex produced upon substrate cleavage increased with the severity of the point mutation. A condensation assay was developed to directly measure fructose 1,6-bisphosphate synthesized by aldolases in the presence of high triose phosphate concentrations. The maximal rate of aldol condensation of triose phosphates, D-glyceralehyde-3-P and dihydroxyacetone-P, was affected by the point mutations to the same extent as the maximal rate of substrate cleavage. Interpretation of the data is consistent with point mutations in the COOH terminus predominantly affecting the proton exchange with the dihydroxyacetone-P enzymatic complex at the carbanion-enamine step and that this step is probably rate-limiting in the catalytic mechanism of recombinant maize aldolase. The role of the COOH-terminal region in aldolases is thus consistent with a sequence dependent modulation of catalytic activity.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Studies on chimeric fusion proteins of human aldolase isozymes A and B.

Several kinds of fusion proteins between human aldolases A and B were prepared by recombinant DNA technology and their enzymic properties were examined. AB chimeras, which have aldolase A at the N-terminal region and aldolase B at the C-terminal region, were scarcely obtained, while BA chimeras were abundant (Kitajima et al., (1990), J. Biol. Chem., 265, 17493-17498). All the BAB chimeras, aldolase A fragments inserted in aldolase B, showed activity assignable to aldolase B type, which imply an essential role of Tyr residue at the C-terminus of aldolase A in the binding of fructose-1,6-bisphosphate (Fru-1,6-P2). BAB chimeras also showed reactivity to effectors such as fructose-2,6-bisphosphate (Fru-2,6-P2) and pyridoxal 5-phosphate (PLP), in a similar manner to aldolase B. BAB108 has a similarity to the BA108 chimera, but acts differently from other BAB chimeras, suggesting that its structure around active site looks like that of aldolase A.     

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.     

The proton exchange of the pro-S hydrogen atom at C-1 in dihydroxyacetone phosphate and D-fructose 1,6-bisphosphate catalysed by class-I and class-II aldolases.

The efficacy of class-I and class-II aldolases in catalysing the C-1 proton exchange in fructose 1,6-bisphosphate and dihydroxyacetone phosphate was investigated. The rate of this reaction was at least two orders of magnitude slower in class-II than in the class-I aldolases. It is suggested that this difference reflects the formation of different intermediates in the reactions catalysed by the two classes of aldolase.     

Activity and specificity of human aldolases

The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded beta-barrel type structure. The C-terminal "tail" extends from the rim of the beta-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.     

Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.     

Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster

Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.     

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.