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

Biochemistry
R J SpreitzerP D McLaughlin

Abstract

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 hav...Continue Reading

Citations

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