title: Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate.
abstract: Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism.
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina. Electronic address: tronconi@cefobi-conicet.gov.ar.
authors.forename: Marcos Ariel
authors.initials: MA
authors.lastname: Tronconi
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: Mariel Claudia Gerrard
authors.initials: MC
authors.lastname: Wheeler
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: Andrea
authors.initials: A
authors.lastname: Martinatto
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Zubimendi
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: Carlos Santiago
authors.initials: CS
authors.lastname: Andreo
authors.affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
authors.forename: María Fabiana
authors.initials: MF
authors.lastname: Drincovich
mesh_terms: D000495:Allosteric Site; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D005650:Fumarates / Q000494:pharmacology*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000187:drug effects; D008293:Malates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism
chemical_list: D005650:Fumarates; D008293:Malates; D009243:NAD; C030298:malic acid; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); C030574:malate dehydrogenase (decarboxylating)
keywords: Arabidopsis thaliana; Cruciferae; Fumarate; Malic enzyme; Mitochondria; Regulatory site; l-Malate