title: Complementing substitutions at the bottom of the barrel influence catalysis and stability of ribulose-bisphosphate carboxylase/oxygenase.
abstract: The temperature-conditional photosynthesis-deficient mutant 68-4PP of Chlamydomonas reinhardtii results from a Leu-290 to Phe substitution in the chloroplast-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Although this substitution occurs relatively far from the active site, the mutant enzyme has a reduced ratio of carboxylation to oxygenation in addition to reduced thermal stability in vivo and in vitro. In an attempt to understand the role of this region in catalysis, photosynthesis-competent revertants were selected. Two revertants, named R96-4C and R96-8E, were found to arise from second-site mutations that cause V262L and A222T substitutions, respectively. These intragenic suppressor mutations increase the CO2/O2 specificity and carboxylation Vmax back to wild-type values. Based on the crystal structure of the spinach holoenzyme, Leu-290 is not in van der Waals contact with either Val-262 or Ala-222. However, all three residues are located at the bottom of the alpha/beta-barrel active site and may interact with residues of the nuclear encoded small subunits. It appears that amino acid residues at the interface of large and small subunits can influence both stability and catalysis.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hong
authors.forename: R J
authors.initials: RJ
authors.lastname: Spreitzer
mesh_terms: D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D016296:Mutagenesis; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
chemical_list: D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen