Abstract
The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.
Highlights
NADP-dependent malate dehydrogenase (NADP-malate dehydrogenases (MDHs))1 (EC 1.1.1.82) catalyzes the reduction of oxaloacetate into malate in higher plants
Activation kinetics were followed by measuring NADP-MDH activity as a function of time after addition of thioredoxins reduced either chemically by DTT (Fig. 2) or photosynthetically in a reconstituted light activation system
Involvement of Cysteine 207 in the Activation by Thioredoxins—In previous studies [5, 7], we have shown that the removal of the carboxyl-terminal disulfide of NADP-MDH by mutation of cysteines 365 and/or 377 opened the access to the active site but did not accelerate the slow activation rate of the enzyme which was linked to the presence of the amino-terminal disulfide
Summary
Materials—Restriction endonucleases, DNA modification enzymes, T4 DNA ligase, and T4 DNA polymerase were from Appligene. DEAESephacel and Matrex Red A chromatographic supports were respectively from Pharmacia and Grace-Amicon. Chemicals (from Sigma, Boehringer, or Prolabo) were of analytical grade. Escherichia coli strain XL1 blue (CLONTECH) was used to produce high yields of plasmids and M13 single-stranded DNA. All the other strains and vectors were the same as described in Ref. 9. The bases underlined were introduced to replace the codons (written in bold) initially encoding a cysteine with codons coding for a serine or an alanine
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