Computational redesign of Chlamydomonas reinhardtii photosynthetic fructose-1,6-bisphosphatase from the Calvin-Benson-Bassham cycle improves both stability and catalytic capacities in vitro

Photosynthetic carbon fixation is catalyzed by 11 enzymes in the Calvin-Benson-Bassham cycle, a highly conserved metabolic pathway in the green lineage. Fructose-1,6-bisphosphatase (FBPase) contributes to the regeneration of the substrate of carboxylation. While non-photosynthetic paralogs of FBPases contribute to cytosolic neoglucogenesis in a well-documented manner, chloroplast FBPases specific regulations by phospho- and redox-posttranslational modifications, pH, cofactor binding, and homo-oligomeric exchange is only partially understood. In particular, FBPases from green microalgae are potentially target of a multiplicity of disulfide/dithiol exchanges that appear more complex than all their cytosolic and chloroplastic land-plant or cyanobacterial paralogs. In an attempt to decipher the molecular basis of chloroplast FBPase activities and precise redox regulations, we applied computational tools to extensively redesign its protein coding sequence, testing the simultaneous substitution of >13% of its amino acids as guided by distant homology. Resulting modified proteins in pure recombinant form were significantly more stable than the wild-type starting point, that was prone to denaturation or aggregation. In particular, redesigned FBPase favored the catalytically competent homo-tetrameric state. Thermal denaturation assays revealed that the redesign of FBPase sequence starkly increased in thermostability, in good correlation with an augmented enzymatic activity over long periods of time. By comparison with its wild-type counterpart, computationally redesigned FBPase allowed an extensive structure-function study that uncovered the multiplicity of redox regulations of microalgal paralog, their combined contribution to the assembly of the catalytically competent homo-tetrameric state, and will further facilitate molecular engineering of photosynthesis.