Biochemical characterization of thioredoxin-like 3, a conserved protein in the green lineage

DE BONT, LINDA; DHALLEINE, TIPHAINE; TORRESI, FLORENCIA; DONNAY, NATACHA; MATHIOT, SANDRINE; DIDIERJEAN, CLAUDE; ROUHIER, NICOLAS

Conferencia; 16th International Conference on Reactive Oxygen and Nitrogen Species in Plants; 2024

Thioredoxins (TRX) are ubiquitous proteins regulating the redox state of numerousproteins, in particular in chloroplasts. These small proteins have a particular fold and aCxxC motif comprising two active-site cysteines. A predicted chloroplastic proteinpossessing these characteristics has been automatically annotated as TRX-like but itsfunction is unknown and its predicted fold is more related to thiol peroxidases (i.e.AhpC_TSA2-like domain). It is present in microalgae and land plants including treesand we referred it to as TRX-like3. The recombinant TRX-like3 proteins from Arabidopsisthaliana and Chlamydomonas reinhardtii have been expressed in Escherichia coli andpurified. The structure of AtTRX-like3 has been solved. Like regular TRXs, it is amonomer and the CxxC motif present in the first -helix is surface exposed. However,AtTRX-like3 possesses an elongated N-terminal region and an insertion between theβ3 and β4 strands compared to conventional TRXs. While both TRX-like3 are oxidizedby H2O2, no thiol peroxidase activity has been detected. A reductase activity was notdetected either, using insulin as a substrate. Since the biochemical analyses do not giveinformation about a possible function as a reductase, Chlamydomonas mutants havebeen selected. Preliminary results indicate that Trx-like3 mutants exhibit a growthretardation on a minimal medium under light which was not visible when grown photoautotrophically on rich-media. This opens the way for a deeper functional analysis.