- Dual Activity of Quinolinate Synthase: Triose Phosphate Isomerase and Dehydration Activities Play Together to Form Quinolinate
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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
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- Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population
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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
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- Prebiotic synthesis of aminooxazoline-5′-phosphates in water by oxidative phosphorylation
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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.
- Fernández-García,Grefenstette,Powner
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supporting information
p. 4919 - 4921
(2017/07/11)
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- Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase
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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.
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p. 5928 - 5940
(2013/09/23)
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- Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: Partitioning of reaction intermediates in D2O and activation by phosphite dianion
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Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by 1H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry44, 2610 -2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((kC1)D/(kC2) D = 1.1) than for the wildtype enzyme ((kC1) D/(kC2)D = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-13C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by 1H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-13C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-13C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.
- Malabanan, M. Merced,Go, Maybelle K.,Amyes, Tina L.,Richard, John P.
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p. 5767 - 5779
(2012/05/20)
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- Evolution of the indole alkaloid biosynthesis in the genus Hordeum: Distribution of gramine and DIBOA and isolation of the benzoxazinoid biosynthesis genes from Hordeum lechleri
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Two indole alkaloids with defense related functions are synthesized in the genus Hordeum of the Triticeae. Gramine (3(dimethyl-amino-methyl)-indole) is found in H. spontaneum and in some varieties of H. vulgare, the benzoxazinoid 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) is detected in H. roshevitzii, H. brachyantherum, H. flexuosum, H. lechleri. Biosynthesis of DIBOA and of gramine was found to be mutually exclusive in wild Hordeum species, indicating that there was selection against simultaneous expression of both pathways during evolution. The full set of genes required for DIBOA biosynthesis in H.lechleri was isolated and the respective enzyme functions were analyzed by heterologous expression. The cytochrome P450 genes Bx2-Bx5 demonstrate a monophyletic origin for H. lechleri, Triticum aestivum and Zea mays. HlBx2-HlBx5 share highest homology to the orthologous genes of T. aestivum. In contrast, the branch point enzyme of the DIBOA pathway, the indole-3-glycerol phosphate lyase BX1, might have evolved independently in H. lechleri. In all Hordeum species that synthesize DIBOA, DNA sequences homologous to Bx genes are found. In contrast, these sequences are not detectable in the genomes of H. vulgare and H. spontaneum that do not synthesize benzoxazinoids.
- Gruen, Sebastian,Frey, Monika,Gierl, Alfons
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p. 1264 - 1272
(2007/10/03)
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- Reactivities of some energy rich organic orthophosphates towards transition metal ion oxidants
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The orthophosphates, α- and β-glycerophosphates, react with HCrO-4 in HClO4 medium to give 1:1 intermediates which subsequently decomposes to give products of the reactions.The rate is directly proportional to +> in each reaction.Thermodynamic parameters associated with the equilibrium step and the activation parameters associated with the slowest step has been evaluated for each substrate.An attempt has been made to correlate the results obtained earlier with other transition metal ion oxidants.
- Gupta, Kalyan Kali Sen,Mahapatra, Ambikesh,Sanyal, Ankan
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p. 332 - 334
(2007/10/02)
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