Satagopan, Sriram , Scott, Stephanie , Tabita, F. Robert .
Identification of a suppressor-mutant substitution in Synechococcus RubisCO that improves the stability of the wild-type enzyme and identifies a hydrophobic pocket close to active site.
Random mutagenesis of the Synechococcus PCC6301 RubisCO large-subunit gene and trans-complementation in a Rhodobacter capsulatus RubisCO deletion strain (SBI-II-) had previously identified a deleterious mutation. Whereas the wild-type rbcLS genes could complement strain SBI-II- for photoautotrophic growth under anaerobic conditions in the presence of 5% CO2, the rbcL-D103V mutant failed to support growth under identical conditions due to an increased Km(CO2). Further random mutagenesis and biological selection was carried out to select for suppressor mutations. A substitution near the active site restored the growth defect by bringing the Km(CO2) value back to that of the wild type. Structural analysis indicated that D103, a surface residue, forms a hydrogen bond with S367 of a neighboring large subunit in the holoenzyme, an interaction that appears to be conserved among hexadecameric RubisCO forms. The residue S367 possibly transmits the structural changes to the active site via changes in the secondary structure that is caused by the loss of the hydrogen bond. The effect of the suppressor-mutant substitution was analyzed in the presence or absence of the D103V substitution. Besides other changes in kinetic properties, the mutant enzyme appeared to have opposing changes in the Km values for CO2 and O2. Structurally, the substitution appeared to also cause major conformational changes as evidenced by the improved thermal stability of the mutant enzyme. The suppressor-mutant substitution occurs in a highly-conserved hydrophobic pocket, which could serve as a potential target for future genetic engineering of RubisCO.
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1 - Ohio State University, Department of Microbiology, 484 W 12th Ave, Columbus, OH, 43210, USA
2 - Ohio State University, Department of Microbiology
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM