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

Hyeoncheol Francis Son,Gyoo Yeol Jung,Kyung-Jin Kim,Tae Hyeon Yoo,Sunghoon Park

doi:10.1038/srep46005

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.

Highlights

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide
On the basis of the docking simulation of 3-HPA binding to the AbKGSADH active site and pertinent biochemical studies, we reveal the structural features of substrate specificity 3-HPA
The overall structure of AbKGSADH shows a conventional conformation for the Aldehyde dehydrogenase (ALDH) fold

Summary

Results and Discussion

AbKGSADH showed less than 10% ALDH activity when NADP+ was used as a cofactor as compared to that when NAD+ was used (Fig. 3a). To elucidate the cofactor binding mode of AbKGSADH, we determined the crystal structure of the protein in complex with NAD+ at a 2.6 Å resolution (Fig. 3b). The aldehyde-group of α-KGSA is stabilized by Gln[160] and Arg[163] through hydrogen bonds, and two catalytic residues, Glu[253] and Cys[287], assist the binding of the molecule (Fig. 4b). The substrate binding pocket is formed by several hydrophobic residues, such as Phe[156], Val[286], Ile[288], Pro[444], and Phe[450], which seem to contribute to the stabilization of the hydrophobic part of α-KGSA (Fig. 4b). Arg[281], a residue that is involved in the stabilization of the 4′-keto-group of α-KGSA, forms a hydrogen bond with the carboxyl-group. Residue number in AbKGSADH AbKGSADH BsDhaS CnGapD4 EcAldH KpPuuC KpYdcW KpYneI ScAld[4]

Hydrophobic pocket

Methods

Additional Information