Structural determinants underlie the essentiality of the m-type thioredoxin TrxA in Synechocystis

Thioredoxins are central regulators of thiol-based redox signaling in photosynthetic organisms, where multiple isoforms with partially overlapping functions coexist. Among them, TrxA is the cyanobacterial homolog of chloroplast m-type thioredoxins and the only thioredoxin known to be essential in cyanobacteria. However, it is unclear whether this essentiality is due to its high intracellular abundance or to its specific molecular properties. Here, we evaluated the ability of different thioredoxins to functionally replace TrxA and used TrxA/TrxB chimeras to identify the structural determinants underlying its functional specificity. Overexpression of TrxB, TrxQ or TrxC did not support complete segregation of a disrupted trxA locus, although the proteins accumulated to levels comparable to those of endogenous TrxA. Similarly, heterologous expression of NTRC did not rescue the phenotype associated with reduced TrxA levels. Comparative structural analysis revealed differences in surface electrostatics and structural features surrounding the catalytic site of TrxA compared with the other thioredoxins. Analysis of TrxB/TrxA chimeras showed that determinants required for functional complementation are primarily located within the C-terminal region of TrxA. Chimeric variants also differed in their ability to activate cyanobacterial FBP/SBPase and pea chloroplast FBPase in vitro, supporting a role for this region in target-specific interactions. Together, these results demonstrate that the essential function of TrxA cannot be explained by protein abundance alone and requires molecular determinants located primarily within its C-terminal region.