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.
Lewis acid mediated regioselective ring opening of benzylglycidol with dibenzyl phosphate: Short and attractive synthesis of dihydroxyacetone phosphate
Meyer, Odile,Ponaire, Sarah,Rohmer, Michel,Grosdemange-Billiard, Catherine
, p. 4347 - 4350 (2006)
(Chemical Equation Presented) A novel, mild, and efficient method was described to introduce a dibenzyl phosphate by ring opening of benzylglycidol mediated by Lewis acids. This methodology was used as a key step for synthesizing the dihydroxyacetone phosphate (DHAP) in only three steps with an overall yield of 74% from the commercially available racemic benzylglycidol.
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.
Enzyme-assisted preparation of isotope-labeled 1-deoxy-D-xylulose 5-phosphate
Hecht,Kis,Eisenreich,Amslinger,Wungsintaweekul,Herz,Rohdich,Bacher
, p. 3948 - 3952 (2001)
Recombinant 1-deoxy-D-xylulose 5-phosphate synthase of Bacillus subtilis was used for the preparation of isotope-labeled 1-deoxy-D-xylulose 5-phosphate using isotope-labeled glucose and/ or isotope-labeled pyruvate as starting materials. The simple one-po
New highly selective inhibitors of class II fructose-1,6-bisphosphate aldolases
Fonvielle, Matthieu,Weber, Philippe,Dabkowska, Kasia,Therisod, Michel
, p. 2923 - 2926 (2004)
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.
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%.
Biosynthesis of isoprenoids. A rapid method for the preparation of isotope-labeled 4-diphosphocytidyl-2C-methyl-D-erythritol
Rohdich,Schuhr,Hecht,Herz,Wungsintaweekul,Eisenreich,Zenk,Bacher
, p. 9571 - 9573 (2000)
4-Diphosphocytidyl-2C-methyl-D-erythritol serves as an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis. The compound has been prepared in millimole quantity by a sequence of one-pot reactions using 13C-labeled pyruvate and dihydroxyacetone phosphate or 13C-labeled glucose as starting materials and recombinant enzymes of the nonmevalonate isoprenoid pathway as catalysts. The method has been used for the preparation of various 4-diphosphocytidyl-2C-methyl-D-erythritol isotopomers in high yield.
