RbcS suppressor mutations improve the thermal stability and CO₂/O₂ specificity of rbcL- mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

Abstract

In the green alga Chlamydomonas reinhardtii, a Leu²⁹⁰-to-Phe (L290F) substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala²²²-to-Thr and Val²⁶²-to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO₂/O₂ specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between β strands A and B of the Rubisco small subunit, which is coded by RbcS1 and RbcS2 nuclear genes, it seemed possible that substitutions in this loop might also suppress L290F. A mutation in a nuclear gene, Rbc-1, was previously found to suppress the biochemical defects of the L290F enzyme at a posttranslational step, but the nature of this gene and its product remains unknown. In the present study, three nuclear-gene suppressors were found to be linked to each other but not to the Rbc-1 locus. DNA sequencing revealed that the RbcS2 genes of these suppressor strains have mutations that cause either Asn⁵⁴-to-Ser or Ala⁵⁷-to-Val substitutions in the small-subunit βA/βB loop. When present in otherwise wild-type cells, with or without the resident RbcS1 gene, the mutant small subunits improve the thermal stability of wild-type Rubisco. These results indicate that the βA/βB loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO₂/O₂ specificity. The small subunit may be a fruitful target for engineering improved Rubisco.