27416-86-0Relevant academic research and scientific papers
Conformational changes in orotidine 5′-monophosphate decarboxylase: A structure-based explanation for how the 5′-phosphate group activates the enzyme
Desai, Bijoy J.,Wood, B. McKay,Fedorov, Alexander A.,Fedorov, Elena V.,Goryanova, Bogdana,Amyes, Tina L.,Richard, John P.,Almo, Steven C.,Gerlt, John A.
, p. 8665 - 8678 (2012)
The binding of a ligand to orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E c?S? complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5′-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5′-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (β/α)8-barrel structure. Our data support a model in which the IBE provided by the 5′-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec?S and Ec?S? complexes relative to the Eo?S complex. The conclusion that the IBE of the 5′-phosphate group provides stabilization to both the E c?S and Ec?S? complexes, not just the Ec?S? complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.
On the observation of discrete fluorine NMR spectra for uridine 5'-β,γ-fluoromethylenetriphosphate diastereomers at basic pH
Hwang, Candy S.,Kashemirov, Boris A.,McKenna, Charles E.
, p. 5315 - 5319 (2014)
Jakeman et al. recently reported the inability to distinguish the diastereomers of uridine 5'-β,γ-fluoromethylenetriphosphate (β,γ-CHF-UTP, 1) by 19F NMR under conditions we previously prescribed for the resolution of the corresponding β,γ-CHF-
UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety
Ginsburg-Shmuel, Tamar,Haas, Michael,Grbic, Djordje,Arguin, Guillaume,Nadel, Yael,Gendron, Fernand-Pierre,Reiser, Georg,Fischer, Bilha
, p. 5483 - 5495 (2012)
P2Y6 nucleotide receptor (P2Y6-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or Pα position with methoxy and/or borano groups, 18-22. Compound 18A, Rp isomer of 5-OMe-UDP(α-B), is the most potent and P2Y6-R selective agonist currently known (EC50 0.008 μM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y2- and P2Y4-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t1/2 = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t1/2 = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y6-R agonist may be of future therapeutic potential.
Product deuterium isotope effect for orotidine 5′-monophosphate decarboxylase: Evidence for the existence of a short-lived carbanion intermediate
Toth, Krisztina,Amyes, Tina L.,Wood, Bryant M.,Chan, Kui,Gerlt, John A.,Richard, John P.
, p. 12946 - 12947 (2007)
A product isotope effect (PIE) of 1.0 was obtained from the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50%) and [6-2H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) H2O/D2O catalyzed by orotidine 5′-monophosphate decarboxylase. This unitary product isotope effect eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. The complete lack of selectivity for the reaction of solvent H and D that is implied by the value of PIE = 1.0 may be enforced by restricted motion of the NL3+ group of the side-chain of Lys-93 that has been proposed to protonate a vinyl carbanion intermediate. Copyright
Rate enhancements brought about by uridine nucleotides in the reduction of NAD+ at the active site of UDP-galactose 4-epimerase
Liu, Yijeng,Arabshahi, Abolfazl,Frey, Perry A.
, p. 29 - 37 (2000)
UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glucose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible oxidation to the 4-keto- glucopyranosyl ring by reaction with the enzyme-bound NAD+. The UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the enzyme that enhances the chemical reactivity of NAD+ toward reducing agents. This is modeled by UMP-dependent reductive inactivation of the epimerase-NAD+ complex by various sugars as well as by borohydrides. The present work shows that UDP also activates the reduction of epimerase-bound NAD+. Furthermore, the reduction of epimerase-NAD+ by glucose at a very slow rate can be observed under anaerobic conditions in the absence of a uridine nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD+ by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of the rate enhancements brought about by UMP and UDP to be estimated. The rate enhancements by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol-1, respectively, in the activation energy. A decrease of 4.0 kcal mol-1 in the activation energy for reduction by NaBH3CN was brought about by UMP-binding. The maximum increases in the reduction potential of epimerase-NAD+ induced by UMP- and UDP-binding are estimated to be 120 and 90 mV, respectively. The results are well correlated with the perturbations of the nicotinamide-13C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220-12230). (C) 2000 Academic Press.
Structural basis for the exceptional stability of bisaminoacylated nucleotides and transfer RNAs
Duca, Maria,Trindle, Carl O.,Hecht, Sidney M.
, p. 11368 - 11377 (2011)
At least one bisaminoacyl-tRNA is synthesized in nature (by Thermus thermophilus phenylalanyl-tRNA synthetase), and many disubstituted tRNAs have been prepared in vitro. Such misacylated tRNAs are able to participate in protein synthesis, even though they lack the free 2-OH group of the 3-terminal adenosine moiety. Their ready participation in protein synthesis implies significant chemical reactivity. The basis for this reactivity has been documented previously. Surprisingly, the aminoacyl moieties of these tRNAs also exhibit exceptional chemical stability. In the present report, bisaminoacylated nucleotides are investigated computationally and experimentally to define the basis for the stability of such species. Molecular modeling of bisalanyl-AMP in the absence of solvent and in the presence of a limited number of water molecules revealed two common features among the low-energy structures. The first was the presence of H-bonding interactions between the two aminoacyl moieties. The second was the presence of a H-bonding interaction between the 2-O-alanyl moiety and the N-3 atom of the adenine nucleobase, typically mediated through a water molecule. The prediction of an interaction between an aminoacyl moiety and the adenine nucleobase was confirmed experimentally by comparing the behavior of bisalanyl-AMP and bisalanyl-UMP in the presence of model nucleophiles. This study suggests a possible role for the adenosine moiety at the 3-end of aminoanyl-tRNAs in controlling the stability and reactivity of the aminoacyl moiety and has important implications for the reactivity and stability of normal aminoacyl-tRNAs.
Conformational changes in orotidine 5′-monophosphate decarboxylase: "remote" residues that stabilize the active conformation
Wood, B. McKay,Amyes, Tina L.,Fedorov, Alexander A.,Fedorov, Elena V.,Shabila, Andrew,Almo, Steven C.,Richard, John P.,Gerlt, John A.
, p. 3514 - 3516 (2010)
The structural factors responsible for the extraordinary rate enhancement (～1017) of the reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and "clamps" the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a "remote" structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases kcat/Km with a minimal effect on kcat, providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5′-phosphate group of orotidine 5′-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion.
Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction: Importance of residues in the orotate binding site
Iiams, Vanessa,Desai, Bijoy J.,Fedorov, Alexander A.,Fedorov, Elena V.,Almo, Steven C.,Gerlt, John A.
, p. 8497 - 8507 (2011)
The reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (k cat/knon = 7.1 ± 1016) and catalytic proficiency [(kcat/KM)/knon = 4.8 ± 1022 M-1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that "substrate destabilization" by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ～5 kcal/mol (contributing ～2.7 ± 103 to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518-5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of kcat by as much as 400-fold but have a minimal effect on the value of kex for exchange of H6 of the FUMP product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease the value of k cat/KM, with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 ± 10 6-fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.
Product deuterium isotope effects for orotidine 5′-monophosphate decarboxylase: Effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate
Toth, Krisztina,Amyes, Tina L.,Wood, Bryant M.,Chan, Kui,Gerlt, John A.,Richard, John P.
, p. 7018 - 7024 (2010)
A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50%) and [6-2H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH2-NL3+ group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.
Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity
Kang, Minhee,Doddapaneni, Kiran,Sarni, Samantha,Heppner, Zach,Wysocki, Vicki,Wu, Zhengrong
, p. 1760 - 1773 (2020)
Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5′-monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi-dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase-like dimeric topology, with each monomer consisting of a five-stranded β-sheet that is sandwiched by five α-helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl-nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2′-hydroxyl of CMP and the ζ-OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.
