Structural and kinetic features of aldehyde dehydrogenase 1A (ALDH1A) subfamily members, cancer stem cell markers active in retinoic acid biosynthesis
Pequerul Pavon, Raquel (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Vera Lillo, Javier (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Giménez Dejoz, Joan (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Crespo, Isidro (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Coines, Joan (Universitat de Barcelona)
Porté, Sergio (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Rovira, Carme (Universitat de Barcelona)
Parés i Casasampera, Xavier (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Farrés, Jaume (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Data:	2020
Resum:	Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.
Ajuts:	Agencia Estatal de Investigación BIO2016-78057
Drets:	Tots els drets reservats.
Llengua:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Matèria:	Aldehyde dehydrogenase ; All-trans-retinaldehyde ; Magnesium ; Retinoic acid ; Substrate-binding pocket ; SDG 3 - Good Health and Well-being
Publicat a:	Archives of biochemistry and biophysics, Vol. 681 (2020) , p. 108256, ISSN 1096-0384

DOI: 10.1016/j.abb.2020.108256
PMID: 31923393

26 p, 1.1 MB

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