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Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Huo L, Davis I, Liu F, Andi B, Esaki S, Iwaki H, Hasegawa Y, Orville AM, Liu A - Nat Commun (2015)

Bottom Line: Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate.We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant.The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA [2] Molecular Basis of Disease Area of Focus Program, Georgia State University, Atlanta, Georgia 30303, USA.

ABSTRACT
Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

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Crystal structures of two distinct catalytic intermediates.(a) Electron density map of the thiohemiacetal intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 30 min. (b) Electron density map of the thioacyl intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 180 min. The 2Fo−Fc electron density map for ligands and Cys302 is contoured to 1.0 σ and shown as a blue mesh. The omit map for ligands and Cys302 is contoured to 2.0 σ and shown as a gray mesh.
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f5: Crystal structures of two distinct catalytic intermediates.(a) Electron density map of the thiohemiacetal intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 30 min. (b) Electron density map of the thioacyl intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 180 min. The 2Fo−Fc electron density map for ligands and Cys302 is contoured to 1.0 σ and shown as a blue mesh. The omit map for ligands and Cys302 is contoured to 2.0 σ and shown as a gray mesh.

Mentions: When E268A-NAD+ crystals are soaked with 2-HMS for 40 min or less, their single-crystal electronic absorption spectra show an absorbance maximum at 422 nm (Supplementary Fig. 5b), as was observed in the solution-state titration and the stopped-flow assays. An individual electronic absorption spectrum for an E268A-NAD+ crystal soaked with 2-HMS for 15 min can be found in Fig. 3e (top). The structure of E268A-NAD+ soaked with 2-HMS for 30 min before flash cooling was solved and refined to 2.15 Å resolution (Fig. 3b). In this structure, a continuous electron density between Cys302-SG and 2-HMS-C6 is observed, similar to the thioacyl intermediate observed in the wild-type enzyme. However, in contrast to the thioacyl intermediate, the density around C6 is less flat, indicating an sp3- rather than sp2-hybridized carbon (Fig. 5a). The angle between the plane of the carbon backbone of the substrate and the formerly aldehydic oxygen is 55±9°, compared with the angle of the wild-type thioacyl intermediate at 26±4° (Supplementary Table 2). More importantly, the C6 of 2-HMS and the C4N of NAD+ are very close (2.4–2.8 Å), making it unlikely that the hydride has been transferred from the substrate. Taken together, these data allow us to assign this intermediate to a thiohemiacetal enzyme adduct (Fig. 3b). A similar intermediate has only been trapped once previously in a crystal that contains no co-substrate22. Hence, this is the first time for this intermediate to be trapped in the presence of NAD+.


Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Huo L, Davis I, Liu F, Andi B, Esaki S, Iwaki H, Hasegawa Y, Orville AM, Liu A - Nat Commun (2015)

Crystal structures of two distinct catalytic intermediates.(a) Electron density map of the thiohemiacetal intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 30 min. (b) Electron density map of the thioacyl intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 180 min. The 2Fo−Fc electron density map for ligands and Cys302 is contoured to 1.0 σ and shown as a blue mesh. The omit map for ligands and Cys302 is contoured to 2.0 σ and shown as a gray mesh.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286809&req=5

f5: Crystal structures of two distinct catalytic intermediates.(a) Electron density map of the thiohemiacetal intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 30 min. (b) Electron density map of the thioacyl intermediate obtained from E268A-NAD+ crystal soaked with 2-HMS for 180 min. The 2Fo−Fc electron density map for ligands and Cys302 is contoured to 1.0 σ and shown as a blue mesh. The omit map for ligands and Cys302 is contoured to 2.0 σ and shown as a gray mesh.
Mentions: When E268A-NAD+ crystals are soaked with 2-HMS for 40 min or less, their single-crystal electronic absorption spectra show an absorbance maximum at 422 nm (Supplementary Fig. 5b), as was observed in the solution-state titration and the stopped-flow assays. An individual electronic absorption spectrum for an E268A-NAD+ crystal soaked with 2-HMS for 15 min can be found in Fig. 3e (top). The structure of E268A-NAD+ soaked with 2-HMS for 30 min before flash cooling was solved and refined to 2.15 Å resolution (Fig. 3b). In this structure, a continuous electron density between Cys302-SG and 2-HMS-C6 is observed, similar to the thioacyl intermediate observed in the wild-type enzyme. However, in contrast to the thioacyl intermediate, the density around C6 is less flat, indicating an sp3- rather than sp2-hybridized carbon (Fig. 5a). The angle between the plane of the carbon backbone of the substrate and the formerly aldehydic oxygen is 55±9°, compared with the angle of the wild-type thioacyl intermediate at 26±4° (Supplementary Table 2). More importantly, the C6 of 2-HMS and the C4N of NAD+ are very close (2.4–2.8 Å), making it unlikely that the hydride has been transferred from the substrate. Taken together, these data allow us to assign this intermediate to a thiohemiacetal enzyme adduct (Fig. 3b). A similar intermediate has only been trapped once previously in a crystal that contains no co-substrate22. Hence, this is the first time for this intermediate to be trapped in the presence of NAD+.

Bottom Line: Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate.We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant.The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA [2] Molecular Basis of Disease Area of Focus Program, Georgia State University, Atlanta, Georgia 30303, USA.

ABSTRACT
Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

Show MeSH
Related in: MedlinePlus