CC-5030 rbcL-AG221 mt+
$30.00
From Robert J. Spreitzer, University of Nebraska, November 2014
Phenotype: requires acetate at 35 °C, temperature-conditional
Using standard methods of directed mutagenesis and chloroplast transformation of rbcL∆-MX3312 mt+ (CC-4696) (Satagopan and Spreitzer 2004), Boon Hoe Lim in Spreitzer’s group created four substitutions (V221C, C256F, K258R, and I265V) together in the Rubisco large subunit. This mutant (also named AG221) represents one of 15 “associated groups” of amino acids that differ between Chlamydomonas and land plants (Du et al. 2003). It was created to investigate phylogenetic differences that influence Rubisco catalysis (Spreitzer et al. 2005). The mutant enzyme has a decrease in CO2/O2 specificity (Lim and Spreitzer, unpublished). The mutant strain can grow on minimal medium at 25 °C, but dies on minimal medium at 35 °C, and has a decreased amount of Rubisco holoenzyme when grown at 35 °C with acetate medium in darkness. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.
Du YC, Peddi SR, Spreitzer RJ (2003) Assessment of structural and functional divergence far from the large subunit active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 278:49401-49405
Satagopan S, Spreitzer RJ (2004) Substitutions at the Asp-473 latch residue of Chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO2/O2 specificity. J Biol Chem 279:14240-14244
Spreitzer RJ, Peddi SR, Satagopan S (2005) Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proc Natl Acad Sci USA 102:17225-17230