57-04-5Relevant articles and documents
Purification and characterization of an allosteric fructose-1,6- bisphosphate aldolase from germinating mung beans (Vigna radiata)
Lal, Ashish,Plaxton, William C.,Kayastha, Arvind M.
, p. 968 - 974 (2005)
Cytosolic fructose-1,6-P2 (FBP) aldolase (ALDc) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 μmol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD c)-IgG. The enzyme's native Mr was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg2+ had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65°C. It demonstrated: (i) a temperature coefficient (Q10) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALDc is bifunctional for FBP and sedoheptulose-1,7-P2 (Km ≈ 17 μM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (Ki values = 2.2, 3.1 and 7.5 mM, respectively). By contrast, the addition of 2 mM ATP: (i) reduced Vmax by about 2-fold, (ii) increased Km(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h = 1.0 and 2.6 in the absence and presence of 2 mM ATP, respectively). Potent feedback inhibition of ALDc by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.
Structure of a class I tagatose-1,6-bisphosphate aldolase: Investigation into an apparent loss of stereospecificity
LowKam, Clotilde,Liotard, Brigitte,Sygusch, Jurgen
, p. 21143 - 21152 (2010)
Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)8 fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys 205, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.
Probing the role of highly conserved residues in triosephosphate isomerase - Analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme
Bandyopadhyay, Debarati,Murthy, Mathur R. N.,Balaram, Hemalatha,Balaram, Padmanabhan
, p. 3863 - 3882 (2015)
Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 C and 45 C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity.
Simple enzymatic in situ generation of dihydroxyacetone phosphate and its use in a cascade reaction for the production of carbohydrates: Increased efficiency by phosphate cycling
Van Herk, Teunie,Hartog, Aloysius F.,Schoemaker, Hans E.,Wever, Ron
, p. 6244 - 6247 (2006)
A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.
Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue
Samanta, Moumita,Murthy,Balaram, Hemalatha,Balaram, Padmanabhan
, p. 1886 - 1896 (2011)
An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues-K12, H95, E97 and E165-are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three-dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000-fold reduction in kcat is observed for E97Q, whereas the E97D mutant shows a 100-fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000-fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 A resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans-to-gauche transition about the Cγ-Cδ (χ3) bond. In the E97D mutant, the K12 side chain maintains the wild-type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.
Synthesis of novel phosphatidyldihydroxyacetone via transphosphatidylation reaction by phospholipase D
Takami,Suzuki
, p. 2136 - 2139 (1994)
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Biosynthesis of terpenoids: Efficient multistep biotransformation procedures affording isotope-labeled 2C-methyl-D-erythritol 4-phosphate using recombinant 2C-methyl-D-erythritol 4-phosphate Synthase
Hecht,Wungsintaweekul,Rohdich,Kis,Radykewicz,Schuhr,Eisenreich,Richter,Bacher
, p. 7770 - 7775 (2001)
This paper describes the recombinant expression of the ispC gene of Escherichia coli specifying 2C-methyl-D-erythritol 4-phosphate synthase in a modified form that can be purified efficiently by metal-chelating chromatography. The enzyme was used for the preparation of isotope-labeled 2C-methyl-D-erythritol 4-phosphate employing isotope-labeled glucose and pyruvate as starting materials. The simple one-pot methods described afford numerous isotopomers of 2C-methyl-D-erythritol 4-phosphate carrying 3H, 13C, or 14C from commercially available precursors. The overall yield based on the respective isotope-labeled starting material is approximately 50%.
Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase
Shi, Rong,McDonald, Laura,Cui, Qizhi,Matte, Allan,Cygler, Miroslaw,Ekiel, Irena
, p. 1302 - 1307 (2011)
The Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (i) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ii) the active site is formed by two subunits, and (iii) the substrate is covalently bound to His218K* of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphorylates the permanently bound ADP coenzyme of DhaL. This phosphoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK-DhaL. The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56K is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56N K with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218K orients the substrate optimally for phosphoryl transfer. Asp109K is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.
Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population
Fenton, Aron W.,Fenton, Kathryn D.,Lamb, Audrey L.,Martin, Tyler A.,Meneely, Kathleen M.,Swint-Kruse, Liskin,Wu, Tiffany
, (2021/11/20)
Some protein positions play special roles in determining the magnitude of protein function: at such “rheostat” positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A (“I98X”). Despite being ~17 ? from the active site and far from subunit interfaces, substitutions at position 98 have rheostatic contributions to the apparent cooperativity (nH) associated with fructose-1,6-bisphosphate substrate binding and moderately affected binding affinity. Next, we crystallized representative I98X variants to assess structural consequences. Residues smaller than the native isoleucine (cysteine and serine) were readily accommodated, and the larger phenylalanine caused only a slight separation of the two parallel helixes. However, the diffraction quality was reduced for I98F, and further reduced for I98Y. Intriguingly, the resolutions of the I98X structures correlated with their nH values. We propose that substitution effects on both nH and crystal lattice disruption arise from changes in the population of aldolase A conformations in solution. In combination with results computed for rheostat positions in other proteins, the results from this study suggest that rheostat positions accommodate a wide range of side chains and that structural consequences manifest as shifted ensemble populations and/or dynamics changes.
Design of Artificial Metabolisms in Layered Nanomaterials for the Enzymatic Synthesis of Phosphorylated Sugars
Mahdi, Rima,Gurard-Hlaine, Christine,Prvot, Vanessa,De Berardinis, Vronique,Forano, Claude,Lemaire, Marielle
, p. 3110 - 3115 (2015/10/19)
Biohybrid nanoreactors operating in one-pot cascade reactions were designed by co-immobilization of enzymes in an inorganic layered matrix, namely, layered double hydroxides. These biohybrid systems were devoted to prepare dihydroxyacetone phosphate (DHAP) and phosphorylated sugars through stereoselective C-C bond formation. In the first system, two kinases were exploited for the in situ generation of DHAP. Increasing the complexity, the second nano-bioreactor combined up to four enzymes to lead to d-fructose-6-phosphate from the aldol-catalyzed addition of dihydroxyacetone to d-glyceraldehyde-3-phosphate generated in situ from DHAP. The biohybrid catalyst showed the same reaction rate as that of the free enzymes and was reusable.
