J Biol Chem, Vol. 274, Issue 49, 34539-34542, December 3, 1999

COMMUNICATION
Direct NMR Observation of the Thioredoxin-mediated Reduction of the Chloroplast NADP-malate Dehydrogenase Provides a Structural Basis for the Relief of Autoinhibition

Isabelle Krimm, Aymeric Goyer^¶, Emmanuelle Issakidis-Bourguet^¶, Myroslawa Miginiac-Maslow^¶, and Jean-Marc Lancelin

From the  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 and the ^¶ Institut de Biotechnologie des Plantes, UMR 8618 CNRS, Bâtiment 630, Université de Paris-Sud, F-91405 Orsay, France

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 Cys³⁶⁵-Cys³⁷⁷ 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 (Ala³⁷⁵ to C-terminal Val³⁸⁹) 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.