title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
authors.forename: Todor
authors.initials: T
authors.lastname: Genkov
authors.forename: Yu-Chun
authors.initials: YC
authors.lastname: Du
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Spreitzer
mesh_terms: 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
chemical_list: D020035:Holoenzymes; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine