title: Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition.
abstract: The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.
authors.affiliation: Laboratoire de RMN Biomoléculaire Associé au CNRS, Université Claude Bernard-Lyon 1 and Ecole Supérieure de Chimie Physique Electronique de Lyon, Bâtiment 308G, F-69622 Villeurbanne, France.
authors.forename: I
authors.initials: I
authors.lastname: Krimm
authors.forename: A
authors.initials: A
authors.lastname: Goyer
authors.forename: E
authors.initials: E
authors.lastname: Issakidis-Bourguet
authors.forename: M
authors.initials: M
authors.lastname: Miginiac-Maslow
authors.forename: J M
authors.initials: JM
authors.lastname: Lancelin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)