Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition

The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its act ivation 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 (ina ctive) 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-dimension al 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 aci d-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an inc reased mobility upon reduction, allowing its direct sequence-specific NMR a ssignment. The location of the flexible peptide in the sequence suggests th at the first part of the C-terminal peptide is still folded near the core o f the enzyme, so that cysteines 365 and 377 remain in proximity to allow fo r an efficient reoxidation/inactivation of the enzyme.