Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type
Résumé
A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRX5) was then investigated.
Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys(175) and Cys(183) (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH - dependent inhibition.
Three cysteine residues (C-145, C-194, C-242) are suggested to have a role in controlling the NADI)* access to the active site, and in stabilizing the NADPH regulatory binding site.
Our results also indicate that the regulatory disulfide involves residues Cys(175) and Cys(183) in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f.
This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic Pl - G6PDH in higher plants. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
