591-57-1Relevant academic research and scientific papers
Chemical and enzymatic methodologies for the synthesis of enantiomerically pure glyceraldehyde 3-phosphates
Gauss, Dominik,Schoenenberger, Bernhard,Wohlgemuth, Roland
, p. 18 - 24 (2014)
Glyceraldehyde 3-phosphates are important intermediates of many central metabolic pathways in a large number of living organisms. d-Glyceraldehyde 3-phosphate (d-GAP) is a key intermediate during glycolysis and can as well be found in a variety of other metabolic pathways. The opposite enantiomer, l-glyceraldehyde 3-phosphate (l-GAP), has been found in a few exciting new pathways. Here, improved syntheses of enantiomerically pure glyceraldehyde 3-phosphates are reported. While d-GAP was synthesized by periodate cleavage of d-fructose 6-phosphate, l-GAP was obtained by enzymatic phosphorylation of l-glyceraldehyde. 1H- and 31P NMR spectroscopy was applied in order to examine pH-dependent behavior of GAP over time and to identify potential degradation products. It was found that GAP is stable in acidic aqueous solution below pH 4. At pH 7, methylglyoxal is formed, whereas under alkaline conditions, the formation of lactic acid could be observed.
Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol
Kim, Taeho,Stogios, Peter J.,Khusnutdinova, Anna N.,Nemr, Kayla,Skarina, Tatiana,Flick, Robert,Joo, Jeong Chan,Mahadevan, Radhakrishnan,Savchenko, Alexei,Yakunin, Alexander F.
, p. 597 - 609 (2020)
Carbon– carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli. The crystal structures of BH1352 and TM1559 at 1.40 –2.50 ? resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.
N-sulfonyl hydroxamate derivatives as inhibitors of class II fructose-1,6-diphosphate aldolase
Gavalda, Sabine,Braga, Remi,Dax, Chantal,Vigroux, Alain,Blonski, Casimir
, p. 5375 - 5377 (2005)
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.
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.
Prebiotic synthesis of aminooxazoline-5′-phosphates in water by oxidative phosphorylation
Fernández-García,Grefenstette,Powner
supporting information, p. 4919 - 4921 (2017/07/11)
RNA is essential to all life on Earth and is the leading candidate for the first biopolymer of life. Aminooxazolines have recently emerged as key prebiotic ribonucleotide precursors, and here we develop a novel strategy for aminooxazoline-5′-phosphate synthesis in water from prebiotic feedstocks. Oxidation of acrolein delivers glycidaldehyde (90%), which directs a regioselective phosphorylation in water and specifically affords 5′-phosphorylated nucleotide precursors in upto 36% yield. We also demonstrated a generational link between proteinogenic amino acids (Met, Glu, Gln) and nucleotide synthesis.
One-pot enzymatic reaction sequence for the syntheses of d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate
Molla, Getachew S.,Wohlgemuth, Roland,Liese, Andreas
, p. 77 - 82 (2015/12/26)
A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.
Dual Activity of Quinolinate Synthase: Triose Phosphate Isomerase and Dehydration Activities Play Together to Form Quinolinate
Reichmann, Debora,Couté, Yohann,Ollagnier De Choudens, Sandrine
, p. 6443 - 6446 (2015/11/09)
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.
Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase
Zhai, Xiang,Amyes, Tina L.,Wierenga, Rik K.,Loria, J. Patrick,Richard, John P.
, p. 5928 - 5940 (2013/09/23)
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.
Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis
Wolfson-Stofko, Brett,Hadi, Timin,Blanchard, John S.
, p. 53 - 61 (2013/11/19)
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.
FSAB: A new fructose-6-phosphate aldolase from Escherichia coli. Cloning, over-expression and comparative kinetic characterization with FSAA
Sanchez-Moreno, Israel,Nauton, Lionel,Thery, Vincent,Pinet, Agnes,Petit, Jean-Louis,De Berardinis, Veronique,Samland, Anne K.,Guerard-Helaine, Christine,Lemaire, Marielle
, p. 9 - 14 (2012/11/07)
Fructose-6-phosphate aldolase B (FSAB) from Escherichia coli was successfully over-expressed as His-tagged recombinant protein. A decameric protein was observed as for FSAA. Unlike FSAA, FSAB is not thermally stable at temperatures higher than 60 °C. The 70% identity between the two aldolases has allowed the generation of a 3D structure which has shown a high similarity of the two active sites. Full kinetic studies towards several substrates have revealed that FSAB catalytic activity is very close to FSAA activity, corroborated by the similarity of their active sites. FSAB has been able to react with three known donors (dihydroxyacetone, hydroxyacetone and glycolaldehyde) but always slightly slower than FSAA.
