Alr2204–Alr2205 form a functional modular thioredoxin system that enhances oxidative stress tolerance in cyanobacteria

The cysteine-mediated catalytic reactions are essential for redox homeostasis in photosynthetic organisms. In the genome of cyanobacterium Anabaena PCC 7120 (Anabaena), the genes encoding thioredoxin reductase (TR; alr2204) and the atypical thioredoxin (Trx; alr2205) are contiguously located, but whether these proteins function as a redox pair remains unknown. Interestingly, Alr2204 and Alr2205, respectively, show high homology to the TR and Trx domain of the chloroplast NADPH-Thioredoxin Reductase C (NTRC). To delve into its possible role, Alr2204 was overexpressed in Anabaena. The resulting strain displayed elevated TR activity and demonstrated enhanced resistance to oxidative stresses. Biochemical characterization showed Alr2204 to be a FAD-associated, predominantly alpha helical protein with NADPH-dependent TR activity. Notably, Alr2204 supported the function of thioredoxin Alr2205, indicating that Alr2204-Alr2205 formed a cognate redox pair wherein Alr2204 donated electrons to Alr2205. When co-expressed in Anabaena, Alr2204 copurified with Alr2205, and together these proteins enhanced the cellular levels of the reduced form of 2-Cys-Prx, a physiologically vital anti-oxidant protein. Importantly, Alr2204-Alr2205 also sustained peroxidase activity of 2-Cys-Prx. This is the first study to show that Alr2204-Alr2205, though independently synthesized, are functionally analogous to the NTRC protein, and thioredoxin systems can be modulated to enhance stress resistance in organisms.