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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.

No MeSH data available.


Active site in the complex of AlDHPyr1147 with isobutyraldehyde (ISO) and NADP+. The active site residues are in green. NADP+ and isobutyraldehyde are in magenta. The 2Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at 1σ and colored blue. (a) The omit Fobs − Fcalc electron density map for the model of the tertiary complex around the region of the substrate. The map is colored green and contoured at 3σ. (b) Positive electron density in the Fobs − Fcalc electron density map for AlDHPyr1147, which is indicative of the possible second conformation of Cys287. The Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at +3.2σ and colored green. Hydrogen bonds are shown by dashed lines.
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fig6: Active site in the complex of AlDHPyr1147 with isobutyraldehyde (ISO) and NADP+. The active site residues are in green. NADP+ and isobutyraldehyde are in magenta. The 2Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at 1σ and colored blue. (a) The omit Fobs − Fcalc electron density map for the model of the tertiary complex around the region of the substrate. The map is colored green and contoured at 3σ. (b) Positive electron density in the Fobs − Fcalc electron density map for AlDHPyr1147, which is indicative of the possible second conformation of Cys287. The Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at +3.2σ and colored green. Hydrogen bonds are shown by dashed lines.

Mentions: On the omit-map calculated for the model of the ternary complex without substrate, there were strong positive densities at the same place in the active sites of all six monomers (A, B, C, D, G, and H). These densities were identified at the places where normally water molecules were observed in the complexes of AIDHPyr1147 with the coenzyme. The densities had similar shapes in different monomers, and the isobutyraldehyde substrate fitted its well. Therefore, we interpreted this density as the substrate isobutyraldehyde (Figure 6(a)). The oxygen atom of the substrate forms hydrogen bonds with the O7N atom of the coenzyme and the OG1 atom of Thr230. The substrate molecule is surrounded by the Ile160, Lys163, Lys164, Phe449, Glu443, and Glu460 residues, the hydrophobic parts of the Lys and Glu side chains turning to the substrate.


NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features
Active site in the complex of AlDHPyr1147 with isobutyraldehyde (ISO) and NADP+. The active site residues are in green. NADP+ and isobutyraldehyde are in magenta. The 2Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at 1σ and colored blue. (a) The omit Fobs − Fcalc electron density map for the model of the tertiary complex around the region of the substrate. The map is colored green and contoured at 3σ. (b) Positive electron density in the Fobs − Fcalc electron density map for AlDHPyr1147, which is indicative of the possible second conformation of Cys287. The Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at +3.2σ and colored green. Hydrogen bonds are shown by dashed lines.
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Related In: Results  -  Collection

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

fig6: Active site in the complex of AlDHPyr1147 with isobutyraldehyde (ISO) and NADP+. The active site residues are in green. NADP+ and isobutyraldehyde are in magenta. The 2Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at 1σ and colored blue. (a) The omit Fobs − Fcalc electron density map for the model of the tertiary complex around the region of the substrate. The map is colored green and contoured at 3σ. (b) Positive electron density in the Fobs − Fcalc electron density map for AlDHPyr1147, which is indicative of the possible second conformation of Cys287. The Fobs − Fcalc electron density map for the AlDHPyr1147 model is contoured at +3.2σ and colored green. Hydrogen bonds are shown by dashed lines.
Mentions: On the omit-map calculated for the model of the ternary complex without substrate, there were strong positive densities at the same place in the active sites of all six monomers (A, B, C, D, G, and H). These densities were identified at the places where normally water molecules were observed in the complexes of AIDHPyr1147 with the coenzyme. The densities had similar shapes in different monomers, and the isobutyraldehyde substrate fitted its well. Therefore, we interpreted this density as the substrate isobutyraldehyde (Figure 6(a)). The oxygen atom of the substrate forms hydrogen bonds with the O7N atom of the coenzyme and the OG1 atom of Thr230. The substrate molecule is surrounded by the Ile160, Lys163, Lys164, Phe449, Glu443, and Glu460 residues, the hydrophobic parts of the Lys and Glu side chains turning to the substrate.

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.