2V63: Crystal Structure Of Rubisco From Chlamydomonas Reinhardtii With A Large-subunit V331a Mutation

Citation:
Abstract
The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, alpha-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O2 specificity.
PDB ID: 2V63Download
MMDB ID: 126779
PDB Deposition Date: 2007/7/13
Updated in MMDB: 2015/02
Experimental Method:
x-ray diffraction
Resolution: 1.8  Å
Source Organism:
Similar Structures:
Biological Unit for 2V63: hexadecameric; determined by author and by software (PQS)
Molecular Components in 2V63
Label Count Molecule
Proteins (16 molecules)
8
Ribulose Bisphosphate Carboxylase Large Chain(Gene symbol: rbcL)
Molecule annotation
8
Ribulose Bisphosphate Carboxylase Small Chain 1(Gene symbol: RBCS1)
Molecule annotation
Chemicals (62 molecules)
1
8
2
8
3
46
* Click molecule labels to explore molecular sequence information.

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