Journal of Biological Chemistry
Volume 289, Issue 31, 1 August 2014, Pages 21433-21450
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Protein Structure and Folding
Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris*

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The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile165 and Met331) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala47) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.

Carbon Fixation
Enzyme Catalysis
Enzyme Structure
Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco)
Site-directed Mutagenesis

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The atomic coordinates and structure factors (codes 4LF1 and 4LF2) have been deposited in the Protein Data Bank (http://wwpdb.org/).

*

This work was supported, in whole or in part, by National Institutes of Health Grant GM095742. This work was also supported by Department of Energy Grant DE-FC02-02ER63421 (to the Protein Expression Technology Center Core Facility at UCLA).

This article contains supplemental Movies S1 and S2.

1

Both authors contributed equally to this work.