Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Drago_Dissertation_edited.pdf (10.16 MB)

ETD Abstract Container

Abstract Header

Microgravity Crystallization and Neutron Diffraction of PLP-Dependent Enzymes

Victoria, Drago Nicole
http://orcid.org/0000-0002-8828-2529
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1651852693785807

Abstract Details

2022, Doctor of Philosophy, University of Toledo, Chemistry.
The metabolically active form of vitamin B6, pyridoxal 5’-phosphate (PLP), is a ubiquitous cofactor present in all branches of life. PLP-dependent enzymes catalyze transamination, racemization, alpha-decarboxylation, aldol cleavage, beta- and gamma-elimination and replacement reactions, and phosphorylation. Due to their significant roles in metabolic pathways, PLP-dependent enzymes are attractive targets for pharmaceuticals, such as antibacterial and anti-tumor agents. Such developments require detailed understanding of PLP-dependent catalytic mechanisms and how PLP is modulated by its host to perform specific chemistry that can only be achieved through atomic-level structural studies. Although X-ray crystallography is the most prevalent technique in structural biology, approximately half the structure is missing in most X-ray protein structures as hydrogens are only resolvable at ultrahigh resolutions. Neutron macromolecular crystallography is a powerful, non-destructive technique to directly determine the positions of hydrogen, or preferentially deuterium. To understand how selective protonation drives reaction specificity in PLP-dependent enzymes, we employ an approach involving X-ray and neutron macromolecular crystallography and subsequent quantum chemical calculations. The primary deterrent of neutron macromolecular crystallography is growing a crystal suitable for neutron diffraction. We have design, tested, and implemented a device for microgravity crystallization aboard the International Space Station for neutron diffraction. With the Toledo Crystallization Box, we were able to demonstrate improved size and neutron diffraction resolution for microgravity-grown perdeuterated tryptophan synthase crystals. Success of TCB implementation on the Center for the Advancement of Science in Space Protein Crystal Growth-15 mission A 2.1 Å neutron diffraction dataset was collected on a microgravity-grown crystal, providing the first neutron structure of TS. Determination of the protonation states in the TS internal aldimine state allow for direct comparison of Fold Type I and Fold Type II internal aldimine structures on a molecular and electronic level. Two neutron structures of aspartate aminotransferase were solved. The 2.2 Å neutron structure of AAT with pyridoxamine 5’-phosphate revealed the direct observation of a low-barrier hydrogen bond between PMP-NSB and catalytic lysine N that preorganizes the active site for the second half-reaction. From the neutron structure, a cluster model was built for density functional theory calculations to inspect the hydrogen bonding interaction, revealing a barrier height to the proton transfer at 1.5 kcal/mol. The neutron structure of AAT reacted with erythro-beta-hydroxyaspartate was solved to 2.2 Å to characterize the stable intermediate state and understand the inhibition of AAT by erythro-beta-hydroxyaspartate. The collective research presented here served to expand the basic knowledge of PLP-dependent catalysis.
Timothy Mueser (Committee Chair)
Andrey Kovalevsky (Committee Member)
John Bellizzi (Committee Member)
Xiche Hu (Committee Member)
247 p.
Biochemistry; Biophysics; Chemistry
microgravity crystallization, neutron diffraction, PLP, PLP-dependent enzymes

Recommended Citations

Citations

  • Victoria, D. N. (2022). Microgravity Crystallization and Neutron Diffraction of PLP-Dependent Enzymes [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1651852693785807

    APA Style (7th edition)

  • Victoria, Drago. Microgravity Crystallization and Neutron Diffraction of PLP-Dependent Enzymes. 2022. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1651852693785807.

    MLA Style (8th edition)

  • Victoria, Drago. "Microgravity Crystallization and Neutron Diffraction of PLP-Dependent Enzymes." Doctoral dissertation, University of Toledo, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1651852693785807

    Chicago Manual of Style (17th edition)

toledo1651852693785807
24
© 2022, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.