- Purification and characterization of an allosteric fructose-1,6- bisphosphate aldolase from germinating mung beans (Vigna radiata)
-
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.
- Lal, Ashish,Plaxton, William C.,Kayastha, Arvind M.
-
-
Read Online
- Structure of a class I tagatose-1,6-bisphosphate aldolase: Investigation into an apparent loss of stereospecificity
-
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.
- LowKam, Clotilde,Liotard, Brigitte,Sygusch, Jurgen
-
-
Read Online
- Probing the role of highly conserved residues in triosephosphate isomerase - Analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme
-
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.
- Bandyopadhyay, Debarati,Murthy, Mathur R. N.,Balaram, Hemalatha,Balaram, Padmanabhan
-
-
Read Online
- Lewis acid mediated regioselective ring opening of benzylglycidol with dibenzyl phosphate: Short and attractive synthesis of dihydroxyacetone phosphate
-
(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.
- Meyer, Odile,Ponaire, Sarah,Rohmer, Michel,Grosdemange-Billiard, Catherine
-
-
Read Online
- 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
-
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.
- Van Herk, Teunie,Hartog, Aloysius F.,Schoemaker, Hans E.,Wever, Ron
-
-
Read Online
- Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue
-
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.
- Samanta, Moumita,Murthy,Balaram, Hemalatha,Balaram, Padmanabhan
-
-
Read Online
- Enzyme-assisted preparation of isotope-labeled 1-deoxy-D-xylulose 5-phosphate
-
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
- Hecht,Kis,Eisenreich,Amslinger,Wungsintaweekul,Herz,Rohdich,Bacher
-
-
Read Online
- 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.
- Fonvielle, Matthieu,Weber, Philippe,Dabkowska, Kasia,Therisod, Michel
-
-
Read Online
- 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
-
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%.
- Hecht,Wungsintaweekul,Rohdich,Kis,Radykewicz,Schuhr,Eisenreich,Richter,Bacher
-
-
Read Online
- Biosynthesis of isoprenoids. A rapid method for the preparation of isotope-labeled 4-diphosphocytidyl-2C-methyl-D-erythritol
-
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.
- Rohdich,Schuhr,Hecht,Herz,Wungsintaweekul,Eisenreich,Zenk,Bacher
-
-
Read Online
- Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase
-
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.
- Shi, Rong,McDonald, Laura,Cui, Qizhi,Matte, Allan,Cygler, Miroslaw,Ekiel, Irena
-
-
Read Online
- Structural characterization of an L-fuculose-1-phosphate aldolase from Klebsiella pneumoniae
-
Fuculose phosphate aldolases play an important role in glycolysis and gluconeogenesis pathways. L-fuculose 1-phosphate aldolase catalyzes the reversible cleavage of L-fuculose 1-phosphate to DHAP and L-lactaldehyde. Class II aldolases found in bacteria are linked to pathogenesis of human pathogens, and have potential applications in the biosynthesis of carbohydrates and other chiral compounds. Here we report the structure of a putative L-fuculose 1-phosphate aldolase (KpFucA) from the nosocomial pathogen Klebsiella pneumoniae to 1.85 ? resolution. The enzyme crystallizes in space group P422 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis confirms that KpFucA is a tetramer in solution. A magnesium ion cofactor and sulfate ion were identified in the active pocket. Enzyme activity assays confirmed that KpFcuA has a strong preference for L-fuculose 1-phosphate as a substrate, but can also catalyze the cleavage of fructose-1,6-bisphosphate and glucose-6-phosphate. This work should provide a starting point for further investigation of the role of KpFucA in K. pneumoniae pathogenesis or in industrial applications.
- Lou, Xiaorui,Zhang, Jianyu,Liu, Shimeng,Wang, Runhao,Li, Weiping,Liu, Ruihua,Zhang, Qionglin,Bartlam, Mark
-
-
- Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population
-
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.
- Fenton, Aron W.,Fenton, Kathryn D.,Lamb, Audrey L.,Martin, Tyler A.,Meneely, Kathleen M.,Swint-Kruse, Liskin,Wu, Tiffany
-
-
- Structural and functional characterization of ydji, an aldolase of unknown specificity in escherichia coli K12
-
The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent kcat of 3.0 s-1. The product, l-glycero-l-galacto-octuluronate-1-phosphate, has a kcat/Km value of 2.1 × 103 M-1 s-1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l-glycero-l-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 ? (Protein Data Bank entry 6OFU), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l-glycero-l-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l-glycero-l-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.
- Huddleston, Jamison P.,Thoden, James B.,Dopkins, Brandon J.,Narindoshvili, Tamari,Fose, Blair J.,Holden, Hazel M.,Raushel, Frank M.
-
p. 3340 - 3353
(2019/08/27)
-
- Facile enzymatic synthesis of ketoses
-
Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step. A spoonful of sugar: A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method, which involves a one-pot mulitenzyme (OPME) reaction, enables the preparation of rare ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.
- Wen, Liuqing,Huang, Kenneth,Wei, Mohui,Meisner, Jeffrey,Liu, Yunpeng,Garner, Kristina,Zang, Lanlan,Wang, Xuan,Li, Xu,Fang, Junqiang,Zhang, Houcheng,Wang, Peng George
-
supporting information
p. 12654 - 12658
(2015/10/28)
-
- Dual Activity of Quinolinate Synthase: Triose Phosphate Isomerase and Dehydration Activities Play Together to Form Quinolinate
-
Quinolinate synthase (NadA) is an Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The capacity of NadA to use G-3P is due to its previously unknown triose phosphate isomerase activity.
- Reichmann, Debora,Couté, Yohann,Ollagnier De Choudens, Sandrine
-
p. 6443 - 6446
(2015/11/09)
-
- Design of Artificial Metabolisms in Layered Nanomaterials for the Enzymatic Synthesis of Phosphorylated Sugars
-
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.
- Mahdi, Rima,Gurard-Hlaine, Christine,Prvot, Vanessa,De Berardinis, Vronique,Forano, Claude,Lemaire, Marielle
-
p. 3110 - 3115
(2015/10/19)
-
- Aldolase-catalyzed synthesis of conformationally constrained iminocyclitols: Preparation of polyhydroxylated benzopyrrolizidines and cyclohexapyrrolizidines
-
A straightforward chemo-enzymatic synthesis of new polyhydroxylated benzopyrrolizidines and cyclohexapyrrolizidines is developed. The two-step strategy consists of l-fuculose-1-phosphate aldolase variant F131A-catalyzed aldol addition of dihydroxyacetone phosphate to rac-N-benzyloxycarbonylindoline- 2-carbaldehyde as well as (2S*,3aS*,7aS*)- and (2S*,3aR*,7aR*)-N-benzyloxycarbonyloctahydroindole-2- carbaldehydes and a subsequent one-step catalytic deprotection-reductive amination.
- Laborda, Pedro,Sayago, Francisco J.,Cativiela, Carlos,Parella, Teodor,Joglar, Jesus,Clapes, Pere
-
supporting information
p. 1422 - 1425
(2014/04/03)
-
- Casuarine stereoisomers from achiral substrates: Chemoenzymatic synthesis and inhibitory properties
-
A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of l-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using d-fructose-6-phosphate aldolase and l-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.
- Concia, Alda Lisa,Gómez, Livia,Parella, Teodor,Joglar, Jesús,Clapés, Pere
-
supporting information
p. 5386 - 5389
(2014/06/23)
-
- Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase
-
Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO 32- complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.
- Zhai, Xiang,Amyes, Tina L.,Wierenga, Rik K.,Loria, J. Patrick,Richard, John P.
-
p. 5928 - 5940
(2013/09/23)
-
- Chemoenzymatic synthesis, structural study and biological activity of novel indolizidine and quinolizidine iminocyclitols
-
The synthesis, conformational study and inhibitory properties of diverse indolizidine and quinolizidine iminocyclitols are described. The compounds were chemo-enzymatically synthesized by two-step aldol addition and reductive amination reactions. The aldol addition of dihydroxyacetone phosphate (DHAP) to N-Cbz-piperidine carbaldehyde derivatives catalyzed by l-rhamnulose 1-phosphate aldolase from Escherichia coli provides the key intermediates. The stereochemical outcome of both aldol addition and reductive amination depended upon the structure of the starting material and intermediates. The combination of both reactions furnished five indolizidine and six quinolizidine type iminocyclitols. A structural analysis by NMR and in silico density functional theory (DFT) calculations allowed us to determine the population of stereoisomers with the trans or cis ring fusion, as a consequence of the inversion of configuration of the bridgehead nitrogen. The trans fusion was by far the most stable, but for certain stereochemical configurations of the 3-hydroxymethyl and hydroxyl substituents both trans and cis fusion stereoisomers coexisted in different proportions. Some of the polyhydroxylated indolizidines and quinolizidines were shown to be moderate to good inhibitors against α-l-rhamnosidase from Penicillium decumbens. Indolizidines were found to be moderate inhibitors of the rat intestinal sucrase and of the exoglucosidase amyloglucosidase from Aspergillus niger. In spite of their activity against α-l-rhamnosidase, all the compounds were ineffective to inhibit the growth of the Mycobacterium tuberculosis, the causative agent of tuberculosis. The Royal Society of Chemistry 2012.
- Gómez, Livia,Garrabou, Xavier,Joglar, Jesús,Bujons, Jordi,Parella, Teodor,Vilaplana, Cristina,Cardona, Pere Joan,Clapés, Pere
-
body text
p. 6309 - 6321
(2012/09/05)
-
- Highly efficient aldol additions of DHA and DHAP to N-Cbz-amino aldehydes catalyzed by l-rhamnulose-1-phosphate and l-fuculose-1-phosphate aldolases in aqueous borate buffer
-
Aldol addition reactions of dihydroxyacetone (DHA) to N-Cbz-amino aldehydes catalyzed by l-rhamnulose-1-phosphate aldolase (RhuA) in the presence of borate buffer are reported. High yields of aldol adduct (e.g. 70-90%) were achieved with excellent (>98:2 syn/anti) stereoselectivity for most S or R configured acceptors, which compares favorably to the reactions performed with DHAP. The stereochemical outcome was different and depended on the N-Cbz-amino aldehyde enantiomer: the S acceptors gave the syn (3R,4S) aldol adduct whereas the R ones gave the anti (3R,4R) diastereomer. Moreover, the tactical use of Cbz protecting group allows simple and efficient elimination of borate and excess of DHA by reverse phase column chromatography or even by simple extraction. This, in addition to the use of unphosphorylated donor nucleophile, makes a useful and expedient methodology for the synthesis of structurally diverse iminocyclitols. The performance of aldol additions of dihydroxyacetone phosphate (DHAP) to N-Cbz-amino aldehydes using RhuA and l-fuculose-1-phosphate aldolase (FucA) catalyst in borate buffer was also evaluated. For FucA catalysts, including FucA F131A, the initial velocity of the aldol addition reactions using DHAP were between 2 and 10 times faster and the yields between 1.5 and 4 times higher than those in triethanolamine buffer. In this case, the retroaldol velocities measured for some aldol adducts were lower than those without borate buffer indicating some trapping effect that could explain the improvement of yields. The Royal Society of Chemistry 2011.
- Garrabou, Xavier,Calveras, Jordi,Joglar, Jesus,Parella, Teodor,Bujons, Jordi,Clapes, Pere
-
body text
p. 8430 - 8436
(2012/05/04)
-
- Dihydroxyacetone phosphate, DHAP, in the crystalline state: monomeric and dimeric forms
-
It was shown that dihydroxyacetone phosphate may exist in both monomeric DHAP (C3H7O6P) and dimeric DHAP-dimer (C6H14O12P2) form. Monomeric DHAP was obtained in the form of four crystalline salts: CaCl(DHAP)·2.9H2O (7a), Ca2Cl3(DHAP)·5H2O (7b), CaCl(DHAP)·2H2O (7c), and CaBr(DHAP)·5H2O (7d) by crystallization from aqueous solutions containing DHAP acid and CaCl2 or CaBr2, or by direct crystallization from a solution containing DHAP precursor and CaCl2. At least one of the salts is stable and may be stored in the crystalline state at room temperature for several months. The dimeric form was obtained by slow saturation of free DHAP syrup with ammonia at -18 °C and isolated in the form of its hydrated diammonium salt (NH4)2(DHAP-dimer)·4H2O (8). The synthesis of the compounds, their crystallization, and crystal structures determined by X-ray crystallography are described. In all 7a-d monomeric DHAP exists in the monoanionic form in an extended (in-plane) cisoid conformation, with both hydroxyl and ester oxygen atoms being synperiplanar to the carbonyl O atom. The crucial structural feature is the coordination manner, in which the terminal phosphate oxygen atoms act as chelating as well as bridging atoms for the calcium cations. Additionally, the DHAP monoanions chelate another Ca2+ by the α-hydroxycarbonyl moiety, in a manner observed previously in dihydroxyacetone (DHA) calcium chloride complexes. In dimeric 8 the anion is a trans isomer with the dioxane ring in a chair conformation with the hydroxyl groups in axial positions and the phosphomethyl group in an equatorial position.
- ?lepokura, Katarzyna,Lis, Tadeusz
-
scheme or table
p. 512 - 529
(2010/04/27)
-
- Activated αβ-unsaturated aldehydes as substrate of dihydroxyacetone phosphate (DHAP)-dependent aldolases in the context of a multienzyme system
-
The utility for carbon-carbon bond formation of a multienzyme system composed of recombinant dihydroxyacetone kinase (DHAK) from Citrobacter freundii, the fructose bisphosphate aldolase from rabbit muscle (RAMA) and acetate kinase (AK) for adenosine triphosphate (ATP) regeneration has been studied. Several aldehydes with great structural diversity, including three α,β-unsaturated aldehydes, have been analysed as acceptor substrates. It was found that α,β-unsaturated aldehydes bearing an electron-withdrawing group in the β position to the double bond with a trans configuration are good acceptors for RAMA in this multienzyme system. The aldol reaction proceeds with excellent D-threo enantioselectivity and the aldol adduct is obtained in good overall yield. The L-threo and D-erythro enantiomers are also accessible from rhamnulose 1-phosphate aldolase (Rha-1PA) and fuculose 1-phosphate aldolase (Fuc-1PA) catalysed reactions, respectively.
- Sanchez-Moreno, Israel,Iturrate, Laura,Doyagueez, A Elisa G.,Martinez, Juan Antonio,Fernandez-Mayoralas, Alfonso,Garcia-Junceda, Eduardo
-
experimental part
p. 2967 - 2975
(2010/03/25)
-
- Separate synthesis and evaluation of glucitol bis-phosphate and mannitol bis-phosphate, as competitive inhibitors of fructose bis-phosphate aldolases
-
We report the first unambiguous syntheses of glucitol-1,6-bis-phosphate and mannitol-1,6-bis-phosphate and their competitive inhibition of various fructose bis-phosphate aldolases.
- Mabiala-Bassiloua, Charles-Gabin,Zwolinska, Magdalena,Therisod, Helene,Sygusch, Jurgen,Therisod, Michel
-
p. 1735 - 1737
(2008/12/21)
-
- An efficient chemoenzymatic route to dihydroxyacetone phosphate from glycidol for the in situ aldolase-mediated synthesis of monosaccharides
-
We report a new two-step procedure that uses inexpensive rac-glycidol to obtain valuable dihydroxyacetone phosphate (DHAP), a building block for the synthesis of monosaccharide analogues.
- Charmantray, Franck,Dellis, Phillipe,Samreth, Soth,Hecquet, Laurence
-
p. 3261 - 3263
(2007/10/03)
-
- Improved straightforward chemical synthesis of dihydroxyacetone phosphate through enzymatic desymmetrization of 2,2-dimethoxypropane-1,3-diol
-
Dihydroxyacetone phosphate (DHAP) was synthesized in high purity and yield in four steps starting from dihydroxyacetone dimer (DHA) (47% overall yield). DHA was converted into 2,2-dimethoxypropane-1,3-diol, which was desymmetrized by acetylation with lipase AK. The alcohol function was phosphorylated to give dibenzyl phosphate ester 4. From 4, two routes were investigated for large-scale synthesis of DHAP. First, acetate hydrolysis was performed prior to hydrogenolysis of the phosphate protective groups. The acetal hydrolysis was finally catalyzed by the phosphate group itself. Second, acetate and acetal hydrolysis were performed in one single step after hydrogenolysis.
- Charmantray, Franck,El Blidi, Lahssen,Gefflaut, Thierry,Hecquet, Laurence,Bolte, Jean,Lemaire, Marielle
-
p. 9310 - 9312
(2007/10/03)
-
- Short and efficient synthesis of a stock material of dihydroxyacetone phosphate from glycidol
-
As enzymatic syntheses are expensive for a large-scale preparation of DHAP, a precursor leading to DHAP was synthesized in three steps starting from (±) glycidol; the stable benzylated stock material afforded by hydrogenolysis DHAP in high purity, which may be used directly without purification in enzymatic aldol synthesis.
- Meyer, Odile,Rohmer, Michel,Grosdemange-Billiard, Catherine
-
p. 7921 - 7923
(2007/10/03)
-
- Carbohydrates from glycerol: An enzymatic four-step, one-pot synthesis
-
A novel one-pot procedure, involving a cascade of four enzymatic steps, for the synthesis of carbohydrates from glycerol and an aldehyde is reported.
- Schoevaart, Rob,Van Rantwijk, Fred,Sheldon, Roger A.
-
p. 2465 - 2466
(2007/10/03)
-
- A novel efficient synthesis of dihydroxyacetone phosphate and bromoacetol phosphate for use in enzymatic aldol syntheses
-
Dihydroxyacetone phospate (DHAP,7) and bromoacetol phospate (BAP,6) were synthesized in four and five steps, respectively, starting from 1,3- dibromoacetone (2). The key step involves desymetrization and ketone protection of 2 to prepare alcohol 3. Phosphorylation of 3 followed by hydrogenolysis and then deprotection of the ketal function afforded 6. A solution of 7 was prepared after treatment of 6 with NaOH. This route allows a short and convenient preparation of DHAP in large scale and high purity for application to the synthesis of sugar derivatives and preparation of BAP for triosephosphate isomerase inhibition.
- Gefflaut, Thierry,Lemaire, Marielle,Valentin, Marie-Lise,Bolte, Jean
-
p. 5920 - 5922
(2007/10/03)
-
- Monofluorophosphonates as Phosphate Mimics in Bioorganic Chemistry: a Comparative Study of CH2-, CHF- and CF2-Phosphonate Analogues of sn-Glycerol-3-phosphate as Substrates for sn-Glycerol-3-phosphate Dehydrogenase
-
The synthesis of the cyclohexylammonium salts of 3-(S),4-dihydroxy-1(R,S)-fluorobutylphosphonic acid 3 and 1,1-difluoro-3-(S),4-dihydroxybutylphosphonic acid 4 is reported; 3 is a better substrate for NADH linked sn-glycerol-3-phosphate dehydrogenase than the difluoromethylenephosphonate 4; a comparative study of the CH2-, CHF- and CF2-phosphonate analogues of sn-glycerol-3-phosphate is reported.
- Nieschalk, Jens,O'Hagan, David
-
p. 719 - 720
(2007/10/02)
-
- Chemoenzymatic carbon-carbon bond formation leading to non-carbohydrate derivative. Stereoselective synthesis of pentamycin C-11-C-16 fragment
-
Chemoenzymatic formation of 8 from an aldehyde 5 and dihydroxyacetone phosphate 6 was achieved by the use of fructose 1,6-diphosphate aldolase as a catalyst. Transformation of 8 to 20, corresponding to pentamycin C-11-C-16 fragment, was accomplished via 18a and 18b by chemical processes.
- Shimagaki,Muneshima,Kubota,Oishi
-
p. 282 - 286
(2007/10/02)
-
- An improved synthesis of dihydroxyacetone phosphate
-
An improved procedure has been developed for the synthesis of dihydroxyacetone phosphate (DHAP). Reaction of 2,5-diethoxy-p-dioxane-2,5-dimethanol (1) and the trivalent phosphorylating reagent dibenzyl-N,N-diethylphosphoramidite (DDP) in the presence of 1,2,4-triazole or tetrazole followed by oxidation with H2O2 gave 2,5-diethoxy-p-dioxane-2,5-dimethanol-O-21-O-51-bis(phosphate) tetrabenzyl ester (2) in 98% yield. Compound 2 was then hydrogenated in the presence of H2-Pd/C to give, after neutralization, 2,5-diethoxy-p-dioxane-2,5-dimethanol-O-21-O-51-bis(phosphate) as a stable trisodium salt (3) in 84% yield. Treatment of 3 with Dowex 50 (H+) generates 2 equivalents of DHAP.
- Pederson,Esker,Wong
-
p. 2643 - 2648
(2007/10/02)
-
- A COMBINED CHEMICAL AND ENZYMATIC PROCEDURE FOR THE SYNTHESIS OF 1-DEOXYNOJIRIMYCIN AND 1-DEOXYMANNOJIRIMYCIN
-
1-Deoxynojirimycin and 1-deoxymannojirimycin have been prepared via fructose diphosphate aldolase catalysed condensation followed by catalytic intramolecular reductive amination.
- Pederson, Richard L.,Kim, Mahn-Joo,Wong, Chi-Huey
-
p. 4645 - 4648
(2007/10/02)
-
- A NOVEL CONVENIENT PREPARATION OF DIHYDROXYACETONE PHOSPHATE AND ITS USE IN ENZYMATIC ALDOL REACTIONS
-
A new preparation of the stable barium salt of 2,5-bis(phosphonooxymethyl)-2,5-diethoxy-1,4-dioxane Ba-2 is described, which by treatment with DOWEX 50 H(1+) gives dihydroxyacetone phosphate (DHAP) in high yield.DHAP prepared by this method was used for aldolase-catalyzed condensations.
- Effenberger, Franz,Straub, Alexander
-
p. 1641 - 1644
(2007/10/02)
-
- Synthesis of Sugars by Aldolase-Catalyzed Condensation Reactions
-
Dihydroxyacetone phosphate was prepared in 200-mmol scale from dihydroxyacetone by two procedures: reaction with phosphorus oxytrichloride and glycerol kinase catalyzed phosphorylation using ATP with in situ regeneration of ATP by phosphoenolpyruvate or acetyl phosphate.Dihydroxyacetone phosphate was converted to fructose 6-phosphate in 80percent yield by exposure to a mixture of co-immobilized triosephosphate isomerase and aldolase followed by acid hydrolysis of the condensation product fructose 1,6-bisphosphate.Fructose 6-phosphate was subsequently converted by chemical and enzymatic schemes into fructose, glucose 6-phosphate, and glucose.Practical procedures are described for the preparation of D- and L-glyceraldehyde 3-phosphate and for several hexoses labeled with 13C in the C-2 and C-2,5 positions.
- Wong, Chi-Huey,Whitesides, George M.
-
p. 3199 - 3205
(2007/10/02)
-