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Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense

View Article: PubMed Central - PubMed

ABSTRACT

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, α-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD+. Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde- and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate.

No MeSH data available.


Substrate binding mode of AbKGSADH.(a) Electrostatic potential surface presentation of substrate binding mode of AbKGSADH. The AbKGSADH structure is shown as an electrostatic potential surface presentation. The binding mode of α-ketoglutarace semialdehyde (α-KGSA), succinate semialdehyde (SSA), and 3-hydroxypropionaldehyde (3-HPA) is predicted by molecular docking simulation. α-KGSA, SSA, and 3-HPA are presented by stick models with grey, cyan, and green colors, respectively. The right figure is rotated 90 degrees vertically from the left figure. (b,c,d) Substrate binding mode of AbKGSADH. The substrate binding mode of α-KGSA (b), SSA (c), and 3-HPA (d) in AbKGSADH. The AbKGSADH structure is shown as a cartoon diagram. The NTD, and the CTD is distinguished with light-blue and orange colors, respectively. The residues involved in the substrate binding are shown as stick models and labeled appropriately. Secondary structure elements are labeled. α-KGSA, SSA, and 3-HPA are shown as stick models with grey, cyan, and green colors, respectively. Hydrogen bonds involved in the substrate binding are shown with red-colored dotted lines. (e) Site-directed mutagenesis of AbKGSADH. Residues involved in binding of the 3-HPA substrate are replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type AbKGSADH.
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f4: Substrate binding mode of AbKGSADH.(a) Electrostatic potential surface presentation of substrate binding mode of AbKGSADH. The AbKGSADH structure is shown as an electrostatic potential surface presentation. The binding mode of α-ketoglutarace semialdehyde (α-KGSA), succinate semialdehyde (SSA), and 3-hydroxypropionaldehyde (3-HPA) is predicted by molecular docking simulation. α-KGSA, SSA, and 3-HPA are presented by stick models with grey, cyan, and green colors, respectively. The right figure is rotated 90 degrees vertically from the left figure. (b,c,d) Substrate binding mode of AbKGSADH. The substrate binding mode of α-KGSA (b), SSA (c), and 3-HPA (d) in AbKGSADH. The AbKGSADH structure is shown as a cartoon diagram. The NTD, and the CTD is distinguished with light-blue and orange colors, respectively. The residues involved in the substrate binding are shown as stick models and labeled appropriately. Secondary structure elements are labeled. α-KGSA, SSA, and 3-HPA are shown as stick models with grey, cyan, and green colors, respectively. Hydrogen bonds involved in the substrate binding are shown with red-colored dotted lines. (e) Site-directed mutagenesis of AbKGSADH. Residues involved in binding of the 3-HPA substrate are replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type AbKGSADH.

Mentions: AbKGSADH is known to utilize both α-ketoglutarate-semialdehyde (α-KGSA) and succinate-semialdehyde (SSA) as substrates27. To elucidate how AbKGSADH accommodates these substrates, we performed molecular docking simulations of AbKGSADH with α-KGSA and SSA. The molecular docking simulations revealed that these two substrates fit well into the somewhat positively charged substrate binding pocket (Fig. 4a). The aldehyde-groups of these substrates, which are the sites of enzyme reaction, are located in the same place around the catalytic residues (Fig. 4a). The aldehyde-group of α-KGSA is stabilized by Gln160 and Arg163 through hydrogen bonds, and two catalytic residues, Glu253 and Cys287, also assist the binding of the molecule (Fig. 4b). The 4′-keto-group of α-KGSA is stabilized by hydrogen bonds with Arg281, and the carboxyl-group of the molecule is stabilized by Glu106 and Gln160. The substrate binding pocket is also formed by several hydrophobic residues, such as Phe156, Val286, Ile288, Pro444, and Phe450, which seem to contribute to the stabilization of the hydrophobic part of α-KGSA (Fig. 4b). The binding of SSA is similar to that of α-KGSA, however, the stabilization of the carboxyl-group of SSA is quite different. Arg281, a residue that is involved in the stabilization of the 4′-keto-group of α-KGSA, forms a hydrogen bond with the carboxyl-group of SSA instead (Fig. 4c). These observations explain how AbKGSADH can accommodate both α-KGSA and SSA as real substrates.


Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense
Substrate binding mode of AbKGSADH.(a) Electrostatic potential surface presentation of substrate binding mode of AbKGSADH. The AbKGSADH structure is shown as an electrostatic potential surface presentation. The binding mode of α-ketoglutarace semialdehyde (α-KGSA), succinate semialdehyde (SSA), and 3-hydroxypropionaldehyde (3-HPA) is predicted by molecular docking simulation. α-KGSA, SSA, and 3-HPA are presented by stick models with grey, cyan, and green colors, respectively. The right figure is rotated 90 degrees vertically from the left figure. (b,c,d) Substrate binding mode of AbKGSADH. The substrate binding mode of α-KGSA (b), SSA (c), and 3-HPA (d) in AbKGSADH. The AbKGSADH structure is shown as a cartoon diagram. The NTD, and the CTD is distinguished with light-blue and orange colors, respectively. The residues involved in the substrate binding are shown as stick models and labeled appropriately. Secondary structure elements are labeled. α-KGSA, SSA, and 3-HPA are shown as stick models with grey, cyan, and green colors, respectively. Hydrogen bonds involved in the substrate binding are shown with red-colored dotted lines. (e) Site-directed mutagenesis of AbKGSADH. Residues involved in binding of the 3-HPA substrate are replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type AbKGSADH.
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f4: Substrate binding mode of AbKGSADH.(a) Electrostatic potential surface presentation of substrate binding mode of AbKGSADH. The AbKGSADH structure is shown as an electrostatic potential surface presentation. The binding mode of α-ketoglutarace semialdehyde (α-KGSA), succinate semialdehyde (SSA), and 3-hydroxypropionaldehyde (3-HPA) is predicted by molecular docking simulation. α-KGSA, SSA, and 3-HPA are presented by stick models with grey, cyan, and green colors, respectively. The right figure is rotated 90 degrees vertically from the left figure. (b,c,d) Substrate binding mode of AbKGSADH. The substrate binding mode of α-KGSA (b), SSA (c), and 3-HPA (d) in AbKGSADH. The AbKGSADH structure is shown as a cartoon diagram. The NTD, and the CTD is distinguished with light-blue and orange colors, respectively. The residues involved in the substrate binding are shown as stick models and labeled appropriately. Secondary structure elements are labeled. α-KGSA, SSA, and 3-HPA are shown as stick models with grey, cyan, and green colors, respectively. Hydrogen bonds involved in the substrate binding are shown with red-colored dotted lines. (e) Site-directed mutagenesis of AbKGSADH. Residues involved in binding of the 3-HPA substrate are replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type AbKGSADH.
Mentions: AbKGSADH is known to utilize both α-ketoglutarate-semialdehyde (α-KGSA) and succinate-semialdehyde (SSA) as substrates27. To elucidate how AbKGSADH accommodates these substrates, we performed molecular docking simulations of AbKGSADH with α-KGSA and SSA. The molecular docking simulations revealed that these two substrates fit well into the somewhat positively charged substrate binding pocket (Fig. 4a). The aldehyde-groups of these substrates, which are the sites of enzyme reaction, are located in the same place around the catalytic residues (Fig. 4a). The aldehyde-group of α-KGSA is stabilized by Gln160 and Arg163 through hydrogen bonds, and two catalytic residues, Glu253 and Cys287, also assist the binding of the molecule (Fig. 4b). The 4′-keto-group of α-KGSA is stabilized by hydrogen bonds with Arg281, and the carboxyl-group of the molecule is stabilized by Glu106 and Gln160. The substrate binding pocket is also formed by several hydrophobic residues, such as Phe156, Val286, Ile288, Pro444, and Phe450, which seem to contribute to the stabilization of the hydrophobic part of α-KGSA (Fig. 4b). The binding of SSA is similar to that of α-KGSA, however, the stabilization of the carboxyl-group of SSA is quite different. Arg281, a residue that is involved in the stabilization of the 4′-keto-group of α-KGSA, forms a hydrogen bond with the carboxyl-group of SSA instead (Fig. 4c). These observations explain how AbKGSADH can accommodate both α-KGSA and SSA as real substrates.

View Article: PubMed Central - PubMed

ABSTRACT

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, α-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD+. Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde- and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate.

No MeSH data available.