Abstract
Thiol-based redox control is one of the important posttranslational mechanisms of the tetrapyrrole biosynthesis pathway. Many enzymes of the pathway have been shown to interact with thioredoxin (TRX) and Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase C (NTRC). We examined the redox-dependency of 5-aminolevulinic acid dehydratase (ALAD), which catalyzed the conjugation of two 5-aminolevulinic acid (ALA) molecules to porphobilinogen. ALAD interacted with TRX f, TRX m and NTRC in chloroplasts. Consequently, less ALAD protein accumulated in the trx f1, ntrc and trx f1/ntrc mutants compared to wild-type control resulting in decreased ALAD activity. In a polyacrylamide gel under non-reducing conditions, ALAD monomers turned out to be present in reduced and two oxidized forms. The reduced and oxidized forms of ALAD differed in their catalytic activity. The addition of TRX stimulated ALAD activity. From our results it was concluded that (i) deficiency of the reducing power mainly affected the in planta stability of ALAD; and (ii) the reduced form of ALAD displayed increased enzymatic activity.
Highlights
Tetrapyrrole biosynthesis (TBS) in higher plants is a tightly controlled metabolic pathway that requires multiple regulatory mechanisms
The 3D-structural analysis and the protein sequence alignment of ALAD1 homologs in the selected plants/photosynthetic organisms (Figure 1) revealed the following peptide domains: a predicted 52-aa N-terminal chloroplast transit peptide, the N-terminus for multimerization and a8-barrel domain including the active site between D220 and Y416
The aminolevulinic acid dehydratase (ALAD) activity of ntrc, trx f1 and ntrc/trx f1 could be increased by DTT by 17%, 24% and 21%, respectively, in comparison to the activity of the mutants without additional DTT
Summary
Tetrapyrrole biosynthesis (TBS) in higher plants is a tightly controlled metabolic pathway that requires multiple regulatory mechanisms. Thiol-based redox-regulation is one of the widespread post-translational mechanisms to modulate the protein activity and stability of stromal proteins involved in various metabolic pathways, like the Calvin–Benson cycle [4,5] or TBS [1]. Thiol-based redox regulation relies on different methods of modification by thiol-disulfide cycling of cysteine residues. These redox-dependent modifications result in the formation of intra- and intermolecular disulfide bonds within a protein and between two proteins, respectively. The reduction of the oxidized thiol groups can be accomplished by redox regulators, such as thioredoxins (TRXs) and reduced Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase C (NTRC). Ten typical TRXs act in the redox regulation of chloroplast
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