Thomas C. Taylor

First Crystal Structure of Rubisco from a Green Alga, Chlamydomonas Reinhardtii.

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 Å resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between β strands A and B of the small subunit (βA-βB loop), which is longer by six amino acid residues than the corresponding region in Rubisco fromSpinacia. Mutations of residues in the βA-βB loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonasstructure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.

Structural transitions during activation and ligand binding in hexadecameric Rubisco inferred from the crystal structure of the activated unliganded spinach enzyme

Activation of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco; EC 4.1.1.39) by CO2 involves carbamylation of Lys 201 and the subsequent binding of a magnesium ion to complete the active site. The refined crystal structure of activated Rubisco shows that the magnesium ligands are Asp 203, Glu 204, the carbamate of Lys 201, and three water molecules. Structural differences between the unactivated and activated forms are minimal. Substrate binding replaces water ligands around the metal and triggers substantial structural changes in loops covering the active site. This leads to a contraction and tightening of the structure of the large subunits with the movements transmitted to and modulated by the small subunits.

Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the enzyme assimilating CO2 in biology. Despite serious efforts, using many different methods, a detailed understanding of activity and regulation in Rubisco still eludes us. New results in X-ray crystallography may provide a structural framework on which to base experimental approaches for more detailed analyses of the function of Rubisco at the molecular level. This article gives a critical review of the field and summarizes recent results from structural studies of Rubisco.

Deformation of Helix C in the Low Temperature L-intermediate of Bacteriorhodopsin

X-ray and electron diffraction studies of specific reaction intermediates, or reaction intermediate analogues, have produced a consistent picture of the structural mechanism of light-driven proton pumping by bacteriorhodopsin. Of central importance within this picture is the structure of the L-intermediate, which follows the retinal all-trans to 13-cis photoisomerization step of the K-intermediate and sets the stage for the primary proton transfer event from the positively charged Schiff base to the negatively charged Asp-85. Here we report the structural changes in bacteriorhodopsin following red light illumination at 150 K. Single crystal microspectrophotometry showed that only the L-intermediate is populated in three-dimensional crystals under these conditions. The experimental difference Fourier electron density map and refined crystallographic structure were consistent with those previously presented (Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Nature 406, 645-648; Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2001) Photochem. Photobiol. 74, 794-804). Based on the refined crystallographic structures, molecular dynamic simulations were used to examine the influence of the conformational change of the protein that is associated with the K-to-L transition on retinal dynamics. Implications regarding the structural mechanism for proton pumping by bacteriorhodopsin are discussed.

A Common Structural Basis for the Inhibition of Ribulose 1,5-Bisphosphate Carboxylase by 4-Carboxyarabinitol 1,5-Bisphosphate and Xylulose 1,5-Bisphosphate

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the carboxylation of ribulose 1,5-bisphosphate. The reaction catalyzed by Rubisco involves several steps, some of which can occur as partial reactions, forming intermediates that can be isolated. Analogues of these intermediates are potent inhibitors of the enzyme. We have studied the interactions with the enzyme of two inhibitors, xylulose 1,5-bisphosphate and 4-carboxyarabinitol 1,5-bisphosphate, by x-ray crystallography. Crystals of the complexes were formed by cocrystallization under activating conditions. In addition, 4-carboxyarabinitol 1,5-bisphosphate was soaked into preformed activated crystals of the enzyme. The result of these experiments was the release of the activating CO2 molecule as well as the metal ion from the active site when the inhibitors bound to the enzyme. Comparison with the structure of an activated complex of the enzyme indicates that the structural basis for the release of the activator groups is a distortion of the metal binding site due to the different geometry of the C-3 hydroxyl of the inhibitors. Both inhibitors induce closure of active site loops despite the inactivated state of the enzyme. Xylulose 1,5-bisphosphate binds in a hydrated form at the active site.