Dataset Information

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

ABSTRACT: Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 ? resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than ?-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.

SUBMITTER: Meyer D

PROVIDER: S-EPMC3390845 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Meyer Danilo D Neumann Piotr P Koers Eline E Sjuts Hanno H Lüdtke Stefan S Sheldrick George M GM Ficner Ralf R Tittmann Kai K

Proceedings of the National Academy of Sciences of the United States of America 20120622 27

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interloc ...[more]

PMID: 22730460

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Project description:Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2-lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate. Variants carrying either an isofunctional (Glu473Asp) or isosteric (Glu473Gln) substitution exhibit a residual catalytic activity of less than 0.1% but accumulate different intermediates at the steady state. Whereas the predecarboxylation intermediate 2-lactyl-ThDP is accumulated in Glu473Asp because of a 3000-fold slower decarboxylation compared to that of the wild-type enzyme, Glu473Gln is not impaired in decarboxylation but generates a long-lived 2-hydroxyethyl-ThDP carbanion/enamine postdecarboxylation intermediate. CD spectroscopic analysis of the protonic and tautomeric equilibria of the cocatalytic aminopyrimidine part of ThDP indicates that an acidic residue is required at position 473 for proper substrate binding. Wild-type PDC and the Glu473Asp variant bind the substrate analogue acetylphosphinate with the same affinity, implying a similar stabilization of the predecarboxylation intermediate analogue on the enzyme, whereas Glu473Gln fails to bind the analogue. The X-ray crystallographic structure of 2-lactyl-ThDP trapped in the decarboxylation-deficient variant Glu473Asp reveals a common stereochemistry of the intermediate C2? stereocenter; however, the scissile C2?-C(carboxylate) bond deviates by ?25-30° from the perpendicular "maximum overlap" orientation relative to the thiazolium ring plane as commonly observed in ThDP enzymes. Because a reactant-state stabilization of the predecarboxylation intermediate can be excluded to account for the slower decarboxylation, the data suggest a strong stereoelectronic effect for the transition state of decarboxylation as supported by additional DFT studies on models. To the best of our knowledge, this is a very rare example in which the magnitude of a stereoelectronic effect could be experimentally estimated for an enzymatic system. Given that variant Glu473Gln is not decarboxylation-deficient, electrostatic stress can be excluded as a driving force for decarboxylation. The apparent dual function of Glu473 further suggests that decarboxylation and protonation of the incipient carbanion are committed and presumably proceed in the same transition state.

Project description:A new series of azomethine-functionalized compounds was synthesized from the condensation of 2-hydroxy-1,3-propanediamine and 2-thienylcarboxaldehydes in the presence of a drying agent. The derivatives were spectroscopically characterized by NMR, LC-MS, UV/Vis, IR and elemental analysis. Variable temperature 1 H-NMR (-60 to +60 °C) was performed to investigate the effect of solvent polarity; the capability of solvent to form H-bond was found to dramatically influencing the tautomerization process of the desired structures. The calculated thermochemical parameters (ΔH298 , ΔG298 and ΔS298 ) at DFT and MP2 levels of theory explained that 3 b exists in equilibrium with two tautomers. The basis of the electronic absorptions was pursued through Time-Dependent Density-Functional Theory (TD-DFT). Analysis of the structural surfaces was inspected and the molecular electrostatic potential (MEP) demonstrated that the three functionalized compounds were relatively analogous in the electronic distributions. Furthermore, the electrophilic and nucleophilic centers lying on the molecular surfaces were probably playing a key-role in stabilizing the compounds through the nonclassical C-H⋅⋅⋅π interactions and hydrogen bonding. The impact of solvent polarity on absorption spectra were investigated via solvatochromic shifts. For instance, compound 3 c displayed a gradual shift of the maximum absorption to the red area when the solvent polarity was increased, recording a 21 nm of bathochromic shift. In contrast, no significant solvent-effect on 3 a and 3 b was observed. The solvation relation was pursued between Gutmann's donicity numbers the experimental λmax ; exhibited almost positive linear performance with a minor oscillation, that ascribe to the possible weak interface between the molecules of solute and designated solvents. The bandgap energy of all products were assessed experimentally using optical absorption spectra following Tauc approach, giving -4.050 (3 a), -3.900 (3 b) and -3.210 (3 c) eV. However, the ΔE were computationally figured out from TD-DFT simulation to be -4.258 (3 a), -4.022 (3 b) and -3.390 (3 c) eV.

Dataset Information

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Publications

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Similar Datasets

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