Redox regulation of chloroplastic G6PDH activity by thioredoxin occurs through structural changes modifying substrate accessibility and cofactor binding.

In chloroplasts, redox regulation of enzyme activities by TRXs (thioredoxins) allows the co-ordination of light/dark metabolisms such as the reductive (so-called Calvin–Benson) pathway and the OPPP (oxidative pentose phosphate pathway). Although the molecular mechanisms underlying the redox regulation of several TRX-regulated enzymes have been investigated in detail, only partial information was available for plastidial G6PDH (glucose-6-phosphate dehydrogenase) catalysing the first and rate-limiting step of the OPPP. In the present study, we investigated changes in catalytic and structural properties undergone by G6PDH1 from Arabidopsis thaliana upon treatment with TRX f1, the most efficient regulator of the enzyme that did not show a stable interaction with its target. We found that the formation of the regulatory disulfide bridge that leads to activation of the enzyme allows better substrate accessibility to the active site and strongly modifies the cofactor-binding properties. Structural modelling and data from biochemical and biophysical studies of site-directed mutant proteins support a mechanism in which the positioning/function of the highly conserved Arg 131 in the cofactor-binding site can be directly influenced by the redox state of the adjacent regulatory disulfide bridge. These findings constitute another example of modifications to catalytic properties of a chloroplastic enzyme upon redox regulation, but by a mechanism unique to G6PDH.