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Studying NAD(P)H cofactor-binding to alcohol dehydrogenases through global analysis of circular dichroism spectra

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Abstract

The initial step in reactions catalyzed by NAD(P)H-dependent alcohol dehydrogenases (ADHs) is the binding of the cofactor to the active site. To study this process, we measured NAD(P)H concentration-dependent circular dichroism (CD) in the presence of purified enzymes (ADH from horse liver, HLADH; ADH-A from Rhodococcus ruber; YGL157w from Saccharomyces cerevisiae) or enzyme-containing whole cell extract (ADH from Lactobacillus brevis, LbADH). We determined the proportions of binding and non-binding NAD(P)H and the associated dissociation constants (Kd) from matrix least-squares global fitting of law of mass action-derived model. Furthermore, the fitting allowed the back calculation of CD spectra corresponding to the cofactor in its bound conformation. With increasing pH and/or increasing ionic strength, we detected an increase in Kd for the NADH·HLADH complex with the shape of the bound cofactor conformation spectrum remaining unaffected. While the bound cofactor spectrum for the ADH-A·NADH complex was similar to that for HLADH, the corresponding spectra obtained for the NADPH-dependent enzymes YGL157w and LbADH exhibited opposite signs of the most prominent band. In comparison to CD spectra calculated on cofactor geometries from the crystal structures at the sTD-DFT level, we found that the sign of the bound cofactor spectrum correlates with the orientation of the nicotinamide ring of the cofactor in the active site. These results demonstrate the usefulness of the global analysis of cofactor titration CD spectra to study the role of cofactor binding and its geometry in ADH catalysis.

Graphical abstract: Studying NAD(P)H cofactor-binding to alcohol dehydrogenases through global analysis of circular dichroism spectra

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Publication details

The article was received on 31 Jul 2018, accepted on 28 Sep 2018 and first published on 28 Sep 2018


Article type: Paper
DOI: 10.1039/C8CP04869J
Citation: Phys. Chem. Chem. Phys., 2018, Advance Article
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    Studying NAD(P)H cofactor-binding to alcohol dehydrogenases through global analysis of circular dichroism spectra

    M. Marolt and S. Lüdeke, Phys. Chem. Chem. Phys., 2018, Advance Article , DOI: 10.1039/C8CP04869J

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