Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase.
In the green alga Chlamydomonas reinhardtii, an L290F substitution in the chloroplast-encoded large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) causes decreases in carboxylation Vmax, CO2/O2 specificity, and thermal stability. Analysis of photosynthesis-competent revertants selected at the 35 degrees C restrictive temperature identified a rare C65S suppressor substitution in the nuclear-encoded small subunit. C65S enhances catalysis and CO2/O2 specificity in the absence of other wild-type small subunits, and restores thermal stability in vivo. C65S, C65A, and C65P mutant strains were created. C65S and C65A enzymes have normal catalysis, but C65P Rubisco, which contains land-plant Pro, has decreases in carboxylation Vmax/Km and CO2/O2 specificity. In contrast to other small-subunit substitutions that affect specificity, Cys-65 contacts the large subunit, and the C65P substitution does not cause a decrease in holoenzyme thermal stability in vivo or in vitro. Further analysis of the C65P protein may identify structural alterations that influence catalysis separate from those that affect stability.
Genkov
Todor
T
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
Du
Yu-Chun
YC
Spreitzer
Robert J
RJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D020035:Holoenzymes; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D013489:Suppression, Genetic
D020035:Holoenzymes; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine