RESUMEN
Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis.
Asunto(s)
Apoenzimas/química , Coenzimas/química , NADP/química , Oxidorreductasas/química , Subunidades de Proteína/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Apoenzimas/genética , Apoenzimas/metabolismo , Coenzimas/metabolismo , Cristalografía por Rayos X , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , TermodinámicaRESUMEN
The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.
Asunto(s)
Mitocondrias/enzimología , Proteínas Mitocondriales/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Transporte de Electrón/fisiología , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Pliegue de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Quinone oxidoreductase (QOR EC1.6.5.5) catalyzes the reduction of quinone to hydroxyquinone using NADPH as a cofactor. Here we present the crystal structure of the ζ-crystallin-like QOR Zta1 from Saccharomycescerevisiae in apo-form at 2.00 Šand complexed with NADPH at 1.59 Šresolution. Zta1 forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain. Upon NADPH binding to the interdomain cleft, the two domains shift towards each other, producing a better fit for NADPH, and tightening substrate binding. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis defined a potential quinone-binding site that determines the stringent substrate specificity. Moreover, multiple-sequence alignment and kinetics assays implied that a single-residue change from Arg in lower organisms to Gly in vertebrates possibly resulted in elevation of enzymatic activity of ζ-crystallin-like QORs throughout evolution.
