Alanine-scanning mutagenesis of the small-subunit beta A-beta B loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: substitution at Arg-71 affects thermal stability and CO2/O2 specificity.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function. In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae. To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes. Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function. Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values. The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity. Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.
Spreitzer
R J
RJ
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, USA. rspreitzer1@unl.edu
Esquivel
M G
MG
Du
Y C
YC
McLaughlin
P D
PD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics*; D002245:Carbon Dioxide / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D010641:Phenotype; D017433:Protein Structure, Secondary / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D014170:Transformation, Genetic
D002245:Carbon Dioxide; D001120:Arginine; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen