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NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features

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ABSTRACT

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

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Temperature dependence of the initial rate of the oxidation of isobutyraldehyde with AlDHPyr1147 corrected for the spontaneous nonenzymatic reaction (■). The reaction was initiated by the addition of 10 μg of the enzyme to the reaction mixture in standard assay. (ο) The initial rate of the spontaneous nonenzymatic reaction.
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fig1: Temperature dependence of the initial rate of the oxidation of isobutyraldehyde with AlDHPyr1147 corrected for the spontaneous nonenzymatic reaction (■). The reaction was initiated by the addition of 10 μg of the enzyme to the reaction mixture in standard assay. (ο) The initial rate of the spontaneous nonenzymatic reaction.

Mentions: The optimum pH range for the oxidation of isobutyraldehyde is 8.5–9.5. The highest rate of isobutyraldehyde oxidation was observed at 75–80°С (Figure 1). However, since the nonenzymatic reaction involving NADP+ and aldehydes becomes significant at these temperatures, the standard assay was developed at 60°С. The Michaelis constant for NADP+ is 20.9 ± 0.1 μM. For comparison, Km for Ct-FDH and of homologous dehydrogenase from S. mutans (SmAlDH) are 2.0 and 24.5 μM, respectively, at 20°С [34, 35]. The value of Km of AlDHPyr1147 for NADP+ falls in the Km range from 1.4 to 210 μМ for NADP-dependent AlDHs (EC 1.2.1.4). Neither NADPH nor NAD+ is an efficient competitive inhibitor. Thus, the addition of 0.08 mМ NADPH to a standard reaction mixture (0.084 μМ enzyme and 0.3 mM NADP+) leads only to a 20% decrease in activity. The addition of 0.3 mM NAD+ under the same conditions does not change the rate of the standard reaction.


NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features
Temperature dependence of the initial rate of the oxidation of isobutyraldehyde with AlDHPyr1147 corrected for the spontaneous nonenzymatic reaction (■). The reaction was initiated by the addition of 10 μg of the enzyme to the reaction mixture in standard assay. (ο) The initial rate of the spontaneous nonenzymatic reaction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121451&req=5

fig1: Temperature dependence of the initial rate of the oxidation of isobutyraldehyde with AlDHPyr1147 corrected for the spontaneous nonenzymatic reaction (■). The reaction was initiated by the addition of 10 μg of the enzyme to the reaction mixture in standard assay. (ο) The initial rate of the spontaneous nonenzymatic reaction.
Mentions: The optimum pH range for the oxidation of isobutyraldehyde is 8.5–9.5. The highest rate of isobutyraldehyde oxidation was observed at 75–80°С (Figure 1). However, since the nonenzymatic reaction involving NADP+ and aldehydes becomes significant at these temperatures, the standard assay was developed at 60°С. The Michaelis constant for NADP+ is 20.9 ± 0.1 μM. For comparison, Km for Ct-FDH and of homologous dehydrogenase from S. mutans (SmAlDH) are 2.0 and 24.5 μM, respectively, at 20°С [34, 35]. The value of Km of AlDHPyr1147 for NADP+ falls in the Km range from 1.4 to 210 μМ for NADP-dependent AlDHs (EC 1.2.1.4). Neither NADPH nor NAD+ is an efficient competitive inhibitor. Thus, the addition of 0.08 mМ NADPH to a standard reaction mixture (0.084 μМ enzyme and 0.3 mM NADP+) leads only to a 20% decrease in activity. The addition of 0.3 mM NAD+ under the same conditions does not change the rate of the standard reaction.

View Article: PubMed Central - PubMed

ABSTRACT

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

No MeSH data available.


Related in: MedlinePlus