Structural and kinetic features of three human aldehyde dehydrogenases, aldh1a1, aldh1a2 and aldh1a3, active in retinoic acid biosynthesis
- Autores: Raquel Pequerul Pavón
- Directores de la Tesis: Sergio Porté Orduna (dir. tes.), Xavier Parés i Casasampera (dir. tes.), Jaume Farrés Vicén (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Consuelo Guerri Sirera (presid.), José Antonio Biosca Vaqué (secret.), Oriol Gallego Moli (voc.)
- Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
- Materias:
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- Resumen
The aldehyde dehydrogenase (ALDH) superfamily comprises a large number of dimeric and tetrameric proteins with a subunit molecular weight of approximately 55 kDa and different subcellular localization (cytoplasm, mitochondria and endoplasmic reticulum). ALDHs include a cluster of evolutionarily related NAD(P)+-dependent enzymes catalyzing the oxidation of a wide spectrum of aldehydic substrates, generated from various endogenous and exogenous precursors, to their corresponding carboxylic acids.
The Thesis dissertation is a part of the structural and functional studies performed by our group on the role of oxidoreductases in retinoid metabolism, where their catalytic constants with retinoids were determined after solubilization with bovine serum albumin and by activity analysis using an HPLC-based methodology. The Thesis aims to perform an exhaustive and robust structural and kinetic analysis on the human enzymes involved in the irreversible oxidation of retinaldehyde to retinoic acid.
The first part deals with a comparison of the substrate-binding pocket of the human ALDH1A enzymes, which exhibited similar topologies and decreasing volumes in their substrate-binding pockets. The three enzymes were subcloned, overexpressed and affinity purified in their soluble and active form. Their enzymatic activity was characterized with alkanals and alkenals as substrates. In terms of kcat/Km values, ALDH1A3 exhibits the lowest values for all substrates, suggesting a moderate role in the physiological oxidation of these aldehydes. The kcat/Km values of ALDH1A1 and ALDH1A2 indicate a potentially major role in the transformation of these substrates with slightly different substrate specificity. In order to measure activity with a physiological substrate of ALDH1As, retinaldehyde, an optimization of the solvent extraction methodology was carried out. From the evaluated methods, extraction with hexane/dioxane/isopropanol was chosen, since it was the most efficient in recovering retinaldehyde and retinoic acid from the activity buffer, with a yield near 100%. The three enzymes were active with two retinaldehyde isomers and followed Michaelis-Menten kinetics, with Km values in the micromolar range. Related to the kcat values, they were higher for the all-trans isomer (ALDH1A2 and ALDH1A3) or similar for the two isomers (ALDH1A1). ALDH1A3 was the best enzyme in terms of catalytic efficiency, followed by ALDH1A2. Moreover, the activity of ALDH1A enzymes with apo-β-carotenals, derived from the eccentric cleavage of β-carotene, was described for the first time.
The second part of this work is centered on the role of specific residues in the kinetic properties of ALDH1A enzymes. We performed site-directed mutagenesis, based on structural differences of selected residues from the substrate-binding pocket. The substitution L114P in ALDH1A1 was selected to make this part of the structure more similar to that of ALDH1A2. Likewise, in ALDH1A2, four contiguous residue changes, N475G, A476V, L477V and N478S were made to mimic the structure of ALDH1A1. In the ALDH1A1 L114P mutant, the Km values for hexanal and citral were increased by 50-100 fold related to those of the wild-type ALDH1A1. Conversely, the mutant ALDH1A2 exhibited a 50-fold decrease in the Km value for citral. In addition, the 5-fold decrease in the kcat value made the catalytic efficiency of mutant ALDH1A2 for citral to become similar to that of wild-type ALDH1A1. In regard to kinetics with retinaldehyde isomers, the mutants did not show significant differences with the respective wild-type forms, thus the mutated residues are not critical for retinaldehyde specificity.
Finally, inhibition studies of ALDH1A enzymes were performed in order to find novel, potent and selective inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3. For the first time, we described some compounds as ALDH1A inhibitors and these preliminary results appear to be very promising to develop new pharmacophores by using structure-based drug design.
