Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 specificity.
BACKGROUND
Ribulose-1,5-bisphosphate is the rate-limiting enzyme in photosynthesis. The catalytic large subunit of the green-algal enzyme from Chlamydomonas reinhardtii is approxiamtely 90% identical to the flowering-plant sequences, although they confer diverse kinetic properties. To identify the regions that may account for species variation in kinetic properties, directed mutagenesis and chloroplast transformation were used to create four amino-acid substitutions in the carboxy terminus of the Chlamydomonas large subunit to mimic the sequence of higher-specificity plant enzymes.


RESULTS
The quadruple-mutant enzyme has a 10% increase in CO2/O2 specificity and a lower carboxylation catalytic efficiency. The mutations do not seem to influence the protein expression, structural stability or the function in vivo.


CONCLUSIONS
Owing to the decreased carboxylation catalytic efficiency, the quadruple-mutant is not a "better" enzyme. Nonetheless, because of its positive influence on specificity, the carboxy terminus, relatively far from the active site, may serve as a target for enzyme improvement via combinatorial approaches.
Satagopan
Sriram
S
Department of Microbiology, Ohio State University, Columbus, OH 43210, USA. satagopan.1@osu.edu
Spreitzer
Robert J
RJ
D019943:Amino Acid Substitution; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002696:Chlamydomonas / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D020035:Holoenzymes; D007700:Kinetics; D050505:Mutant Proteins; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D010641:Phenotype; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
D020035:Holoenzymes; D050505:Mutant Proteins; D021122:Protein Subunits; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen