Complementing substitutions within loop regions 2 and 3 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase.
An acetate-requiring mutant of the green alga Chlamydomonas reinhardtii, named 28-7J, has been recovered using chemical mutagenesis. It lacks ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme, and accumulates only a small amount of the chloroplast-encoded large subunit. Pulse/chase experiments revealed that large subunits and nuclear-encoded small subunits are synthesized at normal rates. Because the mutant strain displayed uniparental inheritance and failed to complement a known chloroplast rbcL gene mutant strain, the 28-7J rbcL gene was cloned and sequenced to identify the new mutation. A single base change was found that causes large-subunit arginine-217 to be replaced by serine. This substitution occurs within alpha-helix 2 of the alpha/beta-barrel active site. When photosynthesis-competent revertants were selected from mutant 28-7J, revertant R14-A was found to contain a second mutation within the rbcL gene. This intragenic suppressor mutation, named S14-A, causes alanine-242 to be replaced by valine within beta-strand 3. Holoenzyme from the R14-A double-mutant strain was found to have a 51% reduction in the CO2/O2 specificity factor, primarily due to a 91% decrease in the Vmax of carboxylation. The Km for ribulose 1,5-bisphosphate was increased 2-fold. Although the mutant substitutions are separated by 24 residues within the primary structure, they are close to each other in the tertiary structure. In fact, the substituted residues are also close to lysine-201, which must be carbamylated and coordinated with Mg2+ to activate the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Thow
G
G
Department of Biochemistry, University of Nebraska, Lincoln 68583-0718.
Zhu
G
G
Spreitzer
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005467:Floxuridine / Q000633:toxicity; D005796:Genes; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010100:Oxygen / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D005467:Floxuridine; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen