Browsing by Author "Olmos, Jose Luis"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Structural Enzymology with X-ray Crystallography Using an X-ray Free Electron Laser
    (2020-12-04) Olmos, Jose Luis; Phillips, Jr., George N
    X-ray free electron lasers (XFELs) are linear accelerators that generate powerful X-rays and have proven fruitful for the structural and biochemical investigations of protein structure, function, dynamics, and kinetics in crystallo. BlaC is a beta-lactamase from Mycobacterium tuberculosis, and the first step in understanding its function was to examine its structure using an XFEL with and without ceftriaxone—a third generation cephalosporin antibiotic. Development of mix-and-inject-serial crystallography (MISC) has expanded the utility of XFELs to include enzymes, protein molecules that catalyze biological reactions; structural results indicated that this approach would be feasible with BlaC. The hydrolysis and degradation of ceftriaxone by BlaC was therefore investigated employing the MISC method. The results expose the process of antibiotic cleavage and inactivation in real time and in near atomic detail, demonstrating that this approach is both versatile and can be applied to key reactions of enzymes and other biological macromolecules. To complement these results, steady state kinetics for the BlaC reaction were also investigated by UV-visible spectroscopy, and catalytic mutants were generated. The rate parameters measured near physiological conditions agree with previously reported values, whereas those measured near the crystallization conditions are concordant with the structural observations in MISC. Additional X-ray structures were elucidated as part of this thesis. First, the cyanophage P-SSM2 encodes a viral ferredoxin (Fd) that reroutes photosynthesis in the planet’s most prevalent phototroph, Prochlorococcus marinus. This viral ferredoxin (P-SSM2 Fd) was crystallized and its structure elucidated by X-ray crystallography to 1.6 Å to demonstrate that its topology is similar to other ferredoxins based on homology alignments. Further, the electrostatic surface was compared to that of other photosynthetic Fds, which may help explain the differences in midpoint reduction potentials between them. Second, peroxins (PEX proteins) are key to the biogenesis and function of peroxisomes that sequester oxidative metabolic reactions in cells. The X-ray crystal structure of a peroxin protein complex of PEX4 and PEX22 from the reference plant, Arabidopsis thaliana, was determined. Finally, adenylate kinase serves as a model for protein dynamics, and structural investigations of this enzyme from two bacterial sources, Methanotorris igneus and Thermus thermophiles, were carried out.
  • Thumbnail Image
    Item
    The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex—Insights Into Ubiquitination at the Peroxisomal Membrane
    (Frontiers Media S.A., 2022) Traver, Melissa S.; Bradford, Sarah E.; Olmos, Jose Luis; Wright, Zachary J.; Miller, Mitchell D.; Xu, Weijun; Phillips, George N.; Bartel, Bonnie
    Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant Arabidopsis thaliana. PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed Arabidopsis PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in E. coli. The fusion was linked via a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. Arabidopsis PEX4 displays the typical UBC structure expected from its sequence. Although Arabidopsis PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other Arabidopsis UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The Arabidopsis PEX4-PEX22 structure also revealed that the residue altered in pex4-1 (P123L), a mutant previously isolated via a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated in vitro UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced in vitro ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.