Biomedical subjects

Thomas C Taylor

Publications and source records attributed to Thomas C Taylor.

5 recordsLinked to original sources

PubMed2005

Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that loop size is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V(max), K(m)(O(2)), and CO(2)/O(2) specificity, the spinach loop causes complementary decreases in carboxylation V(max), K(m)(O(2)), and K(m)(CO(2)) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO(2)/O(2) specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO(2) fixation.

Animals↗

PubMed2005

Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase.

Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 A resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 A Calpha-Calpha) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 A Calpha-Calpha). Structural analysis of the mutant enzymes identified regions (residues 50-72 of the small subunit and residues 161-164 and 259-264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.

Amino Acid Substitution↗

PubMed2004

The Uppsala Electron-Density Server.

The Uppsala Electron Density Server (EDS; http://eds.bmc.uu.se/) is a web-based facility that provides access to electron-density maps and statistics concerning the fit of crystal structures and their maps. Maps are available for approximately 87% of the crystallographic Protein Data Bank (PDB) entries for which structure factors have been deposited and for which straightforward map calculations succeed in reproducing the published R value to within five percentage points. Here, an account is provided of the methods that are used to generate the information contained in the server. Some of the problems that are encountered in the map-generation process as well as some spin-offs of the project are also discussed.

Computational Biology↗

PubMed2003

Calcium supports loop closure but not catalysis in Rubisco.

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses CO(2) assimilation in biology. A prerequisite for catalysis is an activation process, whereby an active site lysine is selectively carbamylated. The carbamyl group is then stablised by a metal ion, which in vivo is Mg(2+). Other divalent metal ions can replace Mg(2+) as activators in vitro, but the nature of the metal ion strongly influences the catalytic activity of the enzyme and has a differential effect on the ratio of the carboxylation reaction and the competing oxygenation reaction. Biochemical studies show that calcium promotes carbamylation but not catalysis. To investigate the role of the metal in catalysis, we have determined two structures of the enzyme complexed with Ca(2+) and the transition state analogue 2-carboxy-D-arbinitol-1,5-bisphosphate (2CABP). One of the complexes was prepared by soaking 2CABP into crystals of the enzyme-Ca(2+)-product complex, while the other was obtained by cocrystallising the enzyme with calcium and 2CABP under activating conditions. The two crystals belong to different space groups, and one was merohedrally twinned. Both complexes show very similar three-dimensional features. The enzyme is carbamylated at Lys201, and requisite loops close over the bound ligands in the active site, shielding them from the solvent in a manner similar to the corresponding complex with Mg(2+). However, there are subtle differences that could explain the particular role of Ca(2+) in these processes. The larger radius of the calcium ion and its reduced Lewis-acid character causes a significant increase in the required proton hop distance between the C3 proton and the carbamate on Lys201 in the calcium complex. This alone could explain the inability of calcium to sustain catalysis in Rubisco. Similar effects are also expected on subsequent proton transfer steps in the catalytic cycle. Here we also discuss the effect of metal substitution on the dynamics of the ligands around the metal ion.

Calcium↗

PubMed2003

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.

Bacterial Proteins↗