RbcS suppressor mutations improve the thermal stability and CO2/O2 specificity of rbcL- mutant ribulose-1,5-bisphosphate carboxylase/oxygenase.
In the green alga Chlamydomonas reinhardtii, a Leu(290)-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 Ala(222)-to-Thr and Val(262)-to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO(2)/O(2) specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between beta 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 Asn(54)-to-Ser or Ala(57)-to-Val substitutions in the small-subunit betaA/betaB 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 betaA/betaB loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO(2)/O(2) specificity. The small subunit may be a fruitful target for engineering improved Rubisco.
Du
Y C
YC
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA.
Hong
S
S
Spreitzer
R J
RJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002245:Carbon Dioxide; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006361:Heating; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010100:Oxygen; D010788:Photosynthesis; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
D010940:Plant Proteins; C054765:rca protein, plant; D002245:Carbon Dioxide; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen