Double duty for a conserved glutamate in pyruvate decarboxylase: evidence of the participation in stereoelectronically controlled decarboxylation and in protonation of the nascent carbanion/enamine intermediate .

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 C2α stereocenter; however, the scissile C2α-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.

Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate.

Transketolase is a prominent thiamin diphosphate-dependent enzyme in sugar metabolism that catalyzes the reversible transfer of a 2-carbon dihydroxyethyl fragment between a donor ketose and an acceptor aldose. The X-ray structures of transketolase from E. coli in a covalent complex with donor ketoses d-xylulose 5-phosphate (X5P) and d-fructose 6-phosphate (F6P) at 1.47 A and 1.65 A resolution reveal significant strain in the tetrahedral cofactor-sugar adducts with a 25-30 degrees out-of-plane distortion of the C2-Calpha bond connecting the substrates' carbonyl with the C2 of the cofactor's thiazolium part. Both intermediates adopt very similar extended conformations in the active site with a perpendicular orientation of the scissile C2-C3 sugar bond relative to the thiazolium ring. The sugar-derived hydroxyl groups of the intermediates form conserved hydrogen bonds with one Asp side chain, with a cluster of His residues and with the N4' of the aminopyrimidine ring of the cofactor. The phosphate moiety is held in place by electrostatic and hydrogen-bonding interactions with Arg, His, and Ser side chains. With the exception of the thiazolium part of the cofactor, no structural changes are observable during intermediate formation indicating that the active site is poised for catalysis. DFT calculations on both X5P-thiamin and X5P-thiazolium models demonstrate that an out-of-plane distortion of the C2-Calpha bond is energetically more favorable than a coplanar bond. The X-ray structure with the acceptor aldose d-ribose 5-phosphate (R5P) noncovalently bound in the active site suggests that the sugar is present in multiple forms: in a strained ring-closed beta-d-furanose form in C2-exo conformation as well as in an extended acyclic aldehyde form, with the reactive C1 aldo function held close to Calpha of the presumably planar carbanion/enamine intermediate. The latter form of R5P may be viewed as a near attack conformation. The R5P binding site overlaps with those of the leaving group moieties of the covalent donor-cofactor adducts, demonstrating that R5P directly competes with the donor-derived products glyceraldehyde 3-phosphate and erythrose 4-phosphate, which are substrates of the reverse reaction, for the same docking site at the active site and reaction with the DHEThDP enamine.

Density functional theory and molecular dynamics investigations on substituted banana-shaped compounds.

Density functional theory (DFT) calculations and molecular dynamics (MD) simulations on the atomic level were performed on three different substituted banana-shaped compounds derived from 1,3-phenylene bis[4-(4-n-hexyloxyphenyliminomethyl)benzoate] (P-6-O-PIMB). The DFT studies were carried out on the isolated molecules, and in the MD simulations clusters were treated with up to 64 monomers. The effect of polar substituents, such as chlorine and the nitro group, on the central 1,3-phenylene unit of banana-shaped compounds was investigated. In particular, flexibility, polarity, electrostatic potential (ESP) group charge distributions, B-factors, bending angles and molecular lengths were considered. The MD results were analysed by trajectories of significant torsion angles as well as order parameters such as radial atom pair distribution functions g(r), orientational correlation functions g(o), diffusion coefficients (D) and root mean square deviations (RMSD) values. The g(r) and g(o) values show that a certain long range order is generated by the introduction of a NO(2) group in the 2-position of the central 1,3-phenylene ring. In contrast, the chlorination at the 4 and 6 positions of the central 1,3-phenylene unit decreases the long range order tendency by its perturbation effect on the conformations in such molecules. Moreover, g(r) and g(o) values, as well as diffusion coefficients, show that in the NO(2) substituted compound the formation of microphase areas is preferred. Finally, the aggregation effect in such compounds was studied in a systematic way by a comparison of the conformational properties of the isolated molecules and the monomers in the clusters.

DFT and MD studies on the influence of the orientation of ester linkage groups in banana-shaped mesogens.

The influence of the direction of ester linkage groups on the structural and electronic properties of five-ring banana-shaped molecules with a central 1,3-phenylene unit has been investigated including hexyloxy and dodecyloxy terminal chains. DFT studies on the B3LYP/6-31G(d) level were performed on the conformational behaviour of the ten isomers in a systematic way. The one- and two-fold potential energy scans show that the flexibility of the wings significantly depends on the orientation of the carboxyl linkage groups. Moreover, the different directions of the carboxyl groups between the aromatic rings cause remarkable changes on the dipole moment and its components of the molecules. These findings are supported by investigations on the global charge pattern of the molecules calculated from electrostatic potential group charges. The bending angle alpha obtained from a simple model for the five-ring bent-core molecules is a characteristic structural parameter which can be correlated with experimental findings. Calculations on the bent-core molecules in an external electric dipole field related to the direction of their polar axis show remarkable effects with respect to the flexibility and polarity of the isomers. First molecular dynamics simulations on the banana-shaped molecules were carried out within the AMBER 7 package. The trajectories of relevant structural parameters support the findings of the DFT studies. The results concerning the structure and polarity revealed from the DFT and MD calculations of the ten isomers can be correlated with data from dielectric measurements and mesophase properties.

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins.

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.

Molecular dynamics studies on the aggregation of Y-shaped fluoroalkanes.

Molecular dynamics (MD) calculations have been performed on the aggregation of clusters with up to 128 Y-shaped perfluoroalkylated molecules of the type C10F20[C7H15]2 (Y-A/128) and C10H20[C7F15]2 (Y-B/128) as well as mixed clusters (Y-A/64+Y-B/64) using the AMBER 5 program. The effect of the segregation tendency of the chemically different parts and the influence of the steric repulsion due to the wedge shape of the molecules on the structure formation have been studied. The results have been analyzed by snapshots, radial atom pair distribution functions, orientational correlation functions as well as diffusion coefficients and are compared with the corresponding findings on clusters of alkanes and perfluoroalkanes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s008940020092y.