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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.


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Different conformations of the coenzyme in the complexes with AlDHPyr1147. Coenzyme is in magenta, and AlDHPyr1147 is in green. (a) The coenzyme is in two conformations in the Holo-1 and Holo-2 complexes. The omit Fobs − Fcalc electron density map for the model Holo-2 around the coenzyme is contoured at 2.7σ and colored green. (b) NADP+ is in the “hydride transfer” conformation in the Holo-3 model. The omit Fobs − Fcalc electron density map for the model Holo-3 around the coenzyme is contoured at 3σ and colored green. ((c) and (d)) The 2Fobs − Fcalc electron density maps for the AlDHPyr1147 model contoured at 1σ and colored blue. Hydrogen bonds are shown by dashed lines. (c) An enlarged view of coenzyme in the “hydride transfer” conformation in the active site of the Holo-1 model. (d) An enlarged view of coenzyme in the “out” conformation in the Holo-1 model.
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fig5: Different conformations of the coenzyme in the complexes with AlDHPyr1147. Coenzyme is in magenta, and AlDHPyr1147 is in green. (a) The coenzyme is in two conformations in the Holo-1 and Holo-2 complexes. The omit Fobs − Fcalc electron density map for the model Holo-2 around the coenzyme is contoured at 2.7σ and colored green. (b) NADP+ is in the “hydride transfer” conformation in the Holo-3 model. The omit Fobs − Fcalc electron density map for the model Holo-3 around the coenzyme is contoured at 3σ and colored green. ((c) and (d)) The 2Fobs − Fcalc electron density maps for the AlDHPyr1147 model contoured at 1σ and colored blue. Hydrogen bonds are shown by dashed lines. (c) An enlarged view of coenzyme in the “hydride transfer” conformation in the active site of the Holo-1 model. (d) An enlarged view of coenzyme in the “out” conformation in the Holo-1 model.

Mentions: In the Holo-1 structure, the coenzyme adopts the “out” and “hydride transfer” conformations with approximately equal occupancies in all subunits except B (Figure 5(a), Table S1). In subunit B, the coenzyme is only in the “hydride transfer” conformation. In all subunits, the nicotinamide ring of the coenzyme is disordered and the nicotinamide part of the coenzyme is stabilized in the “hydride transfer” conformation by a hydrogen bond between the O2D atom and Glu382 and by a stacking interaction between the nicotinamide ribose and Phe384 (Figure 5(c)). In the “out” conformation, the nicotinamide ribose forms hydrogen bonds with Trp154, Asn332, and Gln335 (Figure 5(d)). In the Holo-1 structure, as opposed to the structure of the apo form, the active site residues have multiple conformations (Figure 4(b)). Cys287 and the main chain for residues 254-255 are in double conformations, while Glu253 has only one conformation with partial occupancy. The electron density corresponding to the second possible conformation of Glu253 was too weak and did not allow building it. When the coenzyme is in the “hydride transfer” conformation, the Glu253 residue is disordered, and the Cys287 side chain is directed away from the coenzyme-binding pocket and forms a hydrogen bond with a water molecule (HOH 3230 in subunit А), which, in turn, forms a hydrogen bond with ND2 of Asn155. When the coenzyme is in the “out” conformation, both catalytic residues are directed towards the center of the coenzyme-binding pocket, like in the apo structure, in which the distances between SG of Cys287 and OE1 of Glu253 vary from 3.12 to 3.82 Å in two different subunits. This attests to the presence of a hydrogen bond between these atoms. Like in the apo form, the Glu253 residue forms a hydrogen bond with the nitrogen atom of Gly255.


NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features
Different conformations of the coenzyme in the complexes with AlDHPyr1147. Coenzyme is in magenta, and AlDHPyr1147 is in green. (a) The coenzyme is in two conformations in the Holo-1 and Holo-2 complexes. The omit Fobs − Fcalc electron density map for the model Holo-2 around the coenzyme is contoured at 2.7σ and colored green. (b) NADP+ is in the “hydride transfer” conformation in the Holo-3 model. The omit Fobs − Fcalc electron density map for the model Holo-3 around the coenzyme is contoured at 3σ and colored green. ((c) and (d)) The 2Fobs − Fcalc electron density maps for the AlDHPyr1147 model contoured at 1σ and colored blue. Hydrogen bonds are shown by dashed lines. (c) An enlarged view of coenzyme in the “hydride transfer” conformation in the active site of the Holo-1 model. (d) An enlarged view of coenzyme in the “out” conformation in the Holo-1 model.
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fig5: Different conformations of the coenzyme in the complexes with AlDHPyr1147. Coenzyme is in magenta, and AlDHPyr1147 is in green. (a) The coenzyme is in two conformations in the Holo-1 and Holo-2 complexes. The omit Fobs − Fcalc electron density map for the model Holo-2 around the coenzyme is contoured at 2.7σ and colored green. (b) NADP+ is in the “hydride transfer” conformation in the Holo-3 model. The omit Fobs − Fcalc electron density map for the model Holo-3 around the coenzyme is contoured at 3σ and colored green. ((c) and (d)) The 2Fobs − Fcalc electron density maps for the AlDHPyr1147 model contoured at 1σ and colored blue. Hydrogen bonds are shown by dashed lines. (c) An enlarged view of coenzyme in the “hydride transfer” conformation in the active site of the Holo-1 model. (d) An enlarged view of coenzyme in the “out” conformation in the Holo-1 model.
Mentions: In the Holo-1 structure, the coenzyme adopts the “out” and “hydride transfer” conformations with approximately equal occupancies in all subunits except B (Figure 5(a), Table S1). In subunit B, the coenzyme is only in the “hydride transfer” conformation. In all subunits, the nicotinamide ring of the coenzyme is disordered and the nicotinamide part of the coenzyme is stabilized in the “hydride transfer” conformation by a hydrogen bond between the O2D atom and Glu382 and by a stacking interaction between the nicotinamide ribose and Phe384 (Figure 5(c)). In the “out” conformation, the nicotinamide ribose forms hydrogen bonds with Trp154, Asn332, and Gln335 (Figure 5(d)). In the Holo-1 structure, as opposed to the structure of the apo form, the active site residues have multiple conformations (Figure 4(b)). Cys287 and the main chain for residues 254-255 are in double conformations, while Glu253 has only one conformation with partial occupancy. The electron density corresponding to the second possible conformation of Glu253 was too weak and did not allow building it. When the coenzyme is in the “hydride transfer” conformation, the Glu253 residue is disordered, and the Cys287 side chain is directed away from the coenzyme-binding pocket and forms a hydrogen bond with a water molecule (HOH 3230 in subunit А), which, in turn, forms a hydrogen bond with ND2 of Asn155. When the coenzyme is in the “out” conformation, both catalytic residues are directed towards the center of the coenzyme-binding pocket, like in the apo structure, in which the distances between SG of Cys287 and OE1 of Glu253 vary from 3.12 to 3.82 Å in two different subunits. This attests to the presence of a hydrogen bond between these atoms. Like in the apo form, the Glu253 residue forms a hydrogen bond with the nitrogen atom of Gly255.

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