Crystallization and preliminary X-ray diffraction studies of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus.

Journal ArticleThe homotetrameric holo-D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus has been crystallized in the presence of NADP+ using the hanging-drop vapour-diffusion method. Crystals grew from a solution containing 2-methyl-2,4-pentanediol and magnesium acetate. A native data set has been collected to 2.1 A using synchrotron radiation and cryocooling. Diffraction data have been processed in the orthorhombic system (space group P21212) with unit-cell dimensions a = 136.7, b = 153.3, c = 74.9 A and one tetramer per asymmetric unit

Enzyme Active Site Loop Revealed as Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-kinetics

International audienceStructural motions are key events in enzyme catalysis, as exemplified by the conformational dynamics associated with the cofactor in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases. We previously showed that after the oxidoreduction step, the reduced cofactor must adopt a flipped conformation, which positions the nicotinamide in a conserved cavity that might constitute the exit door for NAD(P)H. However, the molecular basis that make this movement possible is unknown. Based on the pre- and post-flip X-ray structures, targeted molecular dynamic simulations enabled us to identify the E268LGG271 conserved loop that must shift to allow reduced nicotinamide conformational switch. To monitor cofactor movements within the active site, we used an intrinsic fluorescence resonance energy transfer signal between Trp177 and the reduced nicotinamide moiety to kinetically track the flip during the catalytic cycle of retinal dehydrogenase 2 (ALDH1A2). Decreasing loop flexibility by substituting Ala for Gly271 drastically reduced the rate constant associated with this movement that became rate-limiting. We thus propose that the E268LGG271 loop acts as a gatekeeper for cofactor flipping. Similar approaches applied to a CoA-dependent aldehyde dehydrogenase showed that cofactor flipping likely extends to the whole ALDH family, thus bridging the gap between the well-studied chemical steps and a conformational transition essential for catalysis

Enzyme Active Site Loop Revealed as Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-kinetics

International audienceStructural motions are key events in enzyme catalysis, as exemplified by the conformational dynamics associated with the cofactor in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases. We previously showed that after the oxidoreduction step, the reduced cofactor must adopt a flipped conformation, which positions the nicotinamide in a conserved cavity that might constitute the exit door for NAD(P)H. However, the molecular basis that make this movement possible is unknown. Based on the pre- and post-flip X-ray structures, targeted molecular dynamic simulations enabled us to identify the E268LGG271 conserved loop that must shift to allow reduced nicotinamide conformational switch. To monitor cofactor movements within the active site, we used an intrinsic fluorescence resonance energy transfer signal between Trp177 and the reduced nicotinamide moiety to kinetically track the flip during the catalytic cycle of retinal dehydrogenase 2 (ALDH1A2). Decreasing loop flexibility by substituting Ala for Gly271 drastically reduced the rate constant associated with this movement that became rate-limiting. We thus propose that the E268LGG271 loop acts as a gatekeeper for cofactor flipping. Similar approaches applied to a CoA-dependent aldehyde dehydrogenase showed that cofactor flipping likely extends to the whole ALDH family, thus bridging the gap between the well-studied chemical steps and a conformational transition essential for catalysis

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: New coenzyme conformation and relevance to catalysis.

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family