Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.     

Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus

We report the purification and crystallization of phosphoglycerate kinase from Thermus caldophilus (Tca). The enzyme crystallizes in the P2(1)2(1)2(1) space group (cell dimensions a = 65.1, b = 71.3, c = 80.2 A), with one molecule in the asymmetric unit. A complete set of diffraction data was collected from an orthorhombic crystal up to 1.8 A resolution.     

Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase.

We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.     

Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase

Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co(2+), is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase.     

Suicide inactivation of fructose-1,6-bisphosphate aldolase

2-Keto-4,4,4-trifluorobutyl phosphate (HTFP) was prepared from 3,3,3-trifluoropropionic acid. HTFP acts as an irreversible inhibitor of rabbit muscle aldolase: the loss of activity was time dependent and the inactivation followed a pseudo-first-order process. Values of 1.4 mM for the dissociation constant and 2.3 X 10(-2) s-1 for the reaction rate constant were determined. The kinetic constants do not depend on the enzyme concentration. No effect of thiols on the inactivation rate was detected. Only 1-2 mol of fluoride ions was liberated per inactivated subunit, indicative of a low partition ratio. Dihydroxyacetone phosphate protected the enzyme against the inactivation in a competitive manner, and glyceraldehyde 3-phosphate protected as if it formed a condensation product with HTPF. 5,5'-Dithiobis(2-nitrobenzoic acid) thiol titration showed the loss of one very reactive thiol group per enzyme subunit after inactivation. All those observations seem to agree with a suicide substrate inactivation of aldolase by HTPF.     

Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.     

Rapid purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts

A rapid procedure for the purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts is presented which involves two steps; precipitation of bulk protein with polyethylene glycol and partitioning of remaining soluble protein in aqueous two-phase systems. A 94% pure preparation is obtained within 6h with a yield of 19%. A marked difference in the partition behaviour of the aldolase activity from whole leaf tissue suggested that the procedure is less efficient when leaf extract is used as starting material.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - PEG Polyethylene Glycol - SDS Sodium Dodecyl Sulfate     

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

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) was purified 97-fold from a halophilic archaebacterium Haloferax mediterranei, with a specific activity of 2.8. The enzyme was characterized as a Class II aldolase on the basis of its inhibition by EDTA and other metal chelators. The enzyme had a specific requirement for divalent metal Fe(2+) for activity. Sulfhydryl compounds enhanced aldolase activity.     

MJ0400 from Methanocaldococcus jannaschii exhibits fructose-1,6-bisphosphate aldolase activity

The central carbon metabolism is well investigated in bacteria, but this is not the case for archaea. MJ0400-His₆ from Methanocaldococcus jannaschii catalyzes the cleavage of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with a V _max of 33 mU mg⁻¹ and a K _m of 430 μM at 50 °C. MJ0400-His₆ is inhibited competitively by erythrose-4-phosphate with a K _i of 380 μM and displays heat stability with a half-life of c . 1 h at 100 °C. Hence, MJ0400 is the second gene encoding for an FBP aldolase in M. jannaschii . Previously, MJ0400 was shown to act as an 2-amino-3,7-dideoxy- d - threo -hept-6-ulosonic acid synthase. This indicates that MJ0400 is involved in both the carbon metabolism and the shikimate pathway in M. jannaschii .     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Topography and conformational changes of fructose-1,6-bisphosphate aldolase.

Structure of aldolase, its interaction with nucleotides, the path of enzyme reaction and the scheme of range of conformational changes of this enzyme are presented. Retrospectives and perspectives of aldolase topography investigations are included.     

Crystallization of allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data

L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained. The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots. The crystals grown at pH 6.0 belonged to monoclinic space group P2(1), the cell dimensions being a = 54.8 A, b = 138.2A, c = 86.1 A, and beta = 93.3 degrees. These crystals diffract to beyond 2.5 A spacing and are stable on X-ray irradiation.     

Action of cathepsin D on fructose-1,6-bisphosphate aldolase.

Cathepsin D inactivated aldolase at pH values between 4.2 and 5.2; the chloride, sulphate or iodide, but not citrate or acetate, salts of sodium or potassium accelerated the rate of inactivation. Cathepsin D cleaved numerous peptide bonds in the C-terminus of aldolase, but the major site of cleavage in this region was Leu354-Phe355. The most prominent peptide products of hydrolysis were Phe-Ile-Ser-Asn-His-Ala-Tyr and Phe-Ile-Ser-Asn-His. Up to 20 amino acids were removed from the C-terminus of aldolase, but no further degradation of native aldolase was observed. By contrast, extensive degradation of the 40 000-Mr subunit was observed after aldolase was denatured. The cathepsin D-inactivated aldolase cross-reacted with antibodies prepared against native aldolase and had the same thermodynamic stability as native aldolase, demonstrated by differential scanning calorimetry and fluorescence quenching of tryptophan residues. Furthermore, the cathepsin-modified and native forms of aldolase were both resistant to extensive proteolysis by other purified cellular proteinases and lysosomal extracts at pH values of 4.8-8.0.     

New highly selective inhibitors of class II fructose-1,6-bisphosphate aldolases

Phosphoglycolo amidoxime and phosphoglycolo hydrazide, two new derivatives of phosphoglycolic acid, were synthesised and successfully tested as selective competitive inhibitors of class II FBP-aldolases.     

N-Sulfonyl hydroxamate derivatives as inhibitors of class II fructose-1,6-diphosphate aldolase

Dihydroxyacetone-phosphate and phosphonate derivatives were synthesized bearing a N-sulfonyl hydroxamate moiety. The phosphate derivatives represent competitive inhibitors for the class II-FBP aldolase catalyzed reaction, while the phosphonate isosteres are comparatively weaker inhibitors.     

One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.     

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 functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.     

Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity and coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C, distinguished by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9 A. Notably, the coupling energy (DeltaGI) is anticorrelated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for approximately 70% of the energy difference for the ratio of kcat/Km for the two substrates between aldolase A and aldolase B. These nonadditive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.