title: Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/oxygenase.
abstract: A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
authors.forename: Y C
authors.initials: YC
authors.lastname: Du
authors.forename: R J
authors.initials: RJ
authors.lastname: Spreitzer
mesh_terms: D000818:Animals; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D017433:Protein Structure, Secondary; D018724:Spinacia oleracea / Q000201:enzymology; D013489:Suppression, Genetic; D013696:Temperature
chemical_list: C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase; D011495:Histone-Lysine N-Methyltransferase