Alanine-scanning mutagenesis of the small-subunit #beta#A-#beta#B loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase:substitutionat arg-71 affects thermal stability and CO_2/O_2 specificity

摘要

Ribulose- 1,5 -bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and C0_2/0_2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the ctl/3-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 Chlarnvdoinonas 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 #beta#A-#beta#B 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-7 1) failed to block assembly or eliminate function. Only the Arg-7 1 Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68->Ala and Asp-69-> Ala enzymes have lower K_m(CO_2) values, but these improvements are offset by decreases in carboxylation Vmax values. The Arg-71->Ala enzyme has a decreased carboxylation Vmax and increased K_m(C0_2) and K_m(O_2) values, which account for an observed 8% decrease in C0J02 specificity. Despite the fact that Arg-7 1 is more than 20 1k from the large-subunit active site, it is apparent that the small-subunit #beta#A-#beta#B loop region can influence catalytic efficiency and C0_2/0_2 specificity.