title: Complementing amino acid substitutions within loop 6 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase.
abstract: Photosynthesis-deficient mutant 45-3B of the green alga Chlamydomonas reinhardtii contains a chloroplast mutation that causes valine-331 to be replaced by alanine within the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. This amino acid substitution occurs in loop 6 of the alpha/beta-barrel active site, three residues distant from catalytic lysine-334. The mutation reduces the specific activity of the enzyme and also reduces its CO2/O2 specificity factor by 42%, but the amount of holoenzyme is unaffected. In a previous study, an intragenic-suppressor mutation, named S40-9D, was selected that causes threonine-342 to be replaced by isoleucine, thereby increasing the CO2/O2 specificity of the mutant enzyme by 36%. To determine which other residues might be able to complement the original mutation, nine additional genetically independent revertants have now been analyzed. Another intragenic suppressor, represented by mutation S61-2J, causes glycine-344 to be replaced by serine. This change increases the CO2/O2 specificity of the mutant enzyme by 25%. Of the revertants recovered and analyzed, the mutant enzyme was improved only due to true reversion or by intragenic suppression mediated by substitutions at residues 342 or 344. Changes in the physical properties of the two pairs of complementing substitutions indicate that steric effects within loop 6 are responsible for the observed changes in the CO2/O2 specificity of the enzyme.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln 68583.
authors.forename: Z X
authors.initials: ZX
authors.lastname: Chen
authors.forename: W Z
authors.initials: WZ
authors.lastname: Yu
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: R
authors.initials: R
authors.lastname: Diao
authors.forename: R J
authors.initials: RJ
authors.lastname: Spreitzer
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001665:Binding Sites; D002245:Carbon Dioxide; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010100:Oxygen; D010428:Pentosephosphates / Q000737:chemistry; D010788:Photosynthesis; D011485:Protein Binding; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000737:chemistry; D013489:Suppression, Genetic
chemical_list: D000596:Amino Acids; D010428:Pentosephosphates; D013402:Sugar Alcohols; D002245:Carbon Dioxide; C032687:2-carboxyarabinitol 1,5-biphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen