DOI
https://doi.org/10.25772/RP38-VY07
Author ORCID Identifier
0000-0002-2563-3002
Defense Date
2023
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Dr Martin Safo
Abstract
Structural biology has become an indispensable tool for determining the 3D structures of proteins for a comprehensive understanding of their functions on molecular level, and for elucidating binding cavities that can be targeted for structure-based drug discovery. X-ray crystallography, the most widely used structural biology technique was utilized in addition to other techniques to: (1) understand the role of pyridoxal reductase (PDXI) in pyridoxal 5’-phosphate (PLP) homeostasis; (2) Determine whether the putative PLP binding protein, YggS, is involved in PLP homeostasis; (3) Elucidate the molecular basis underlying Agrobacterium receptor VirA pathogenesis; and (4) Elucidate the structural basis of aromatic aldehydes antisickling activities.
PLP is the biologically active form of vitamin B6, serving as a cofactor for over 180 B6 enzymes that are involved in critical biochemical reactions, e.g. amino acid, and neurotransmitter biosynthesis. In prokaryotes, yeasts and plants, PLP is obtained from both de novo and salvage pathways, while only the salvage pathway occurs in humans. Traditionally, the salvage pathway has been described to involve the enzymes, pyridoxal kinase (PLK), pyridoxine 5’-phosphate oxidase (PNPO) and phosphatases that synthesize PLP. Free PLP is regulated by phosphatases, PLK and PNPO. PDXI, with a reductase activity is also known to play an important role in PLP regulation however, only limited information on PDXI structure and function is available. The research objective is to characterize E. coli PDXI with respect to its catalytic conversion of PL to PN, substrate binding specificity, and regulation. PDXI was produced and the crystal structures of the unliganded PDXI, binary PDXI-NADPH, and ternary PDXI-NADPH-PL complexes determined, the first of such structure in the PDXI family of proteins. Biophysical binding studies using isothermal titration calorimetry (ITC) showPDXI binds to NADPH (3.0±0.3 µM) or NADP+ (27.2±1.3 µM) but not B6 vitamers, however, in the presence of NADPH or NADP+, PL or PN binds PDXI, resulting in a KD of 0.3±0.04 µM and 31±0.2 µM, respectively. These observations are consistent with the crystallographic result that showed that PL requires the presence of NADPH to bind PDXI. Kinetic studies show that in the presence of PL and NADPH, the reaction reached fast equilibrium resulting in 96 ± 1.7 % of PL being converted to PN. The crystal structure shows the consensus AKR active site catalytic residue His126 causes steric interaction with PL, explaining why His is replaced by Arg126 in PDXI. Using crystallography in conjunction with other techniques, we provided insight into PDXI catalytic mechanism and substrate specificity.
E. coli YggS is a PLP-binding orphan protein, linked to PLP homeostasis. This project tests the hypotheses that YggS functions to scavenge free PLP from the cell and shuttle it apo-B6 enzymes. The crystal structures of wt YggS and several Lys-to-Ala YggS variants, with and without PLP or PNP have been solved. PLP binds covalently to the active site residue K36. Kinetic studies show that only 15% of the K36 bound PLP was transferred from YggS to apo-B6 enzyme, and at a very slow rate (0.14 ± 0.02 min-1). PLP binds YggS with a very high affinity of ~1 nM, but dissociates from YggS very slowly. These observations are inconsistent with YggS serving as a role for B6 activation, and probably not even that of PLP reservoir in the cell.
Agrobacterium tumefaciens is a plant pathogen that causes billions of dollars in plant damage. A. tumefaciens uses a transmembrane histidine kinase (VirA) to initiate pathogenesis by enabling VirA to respond to signal upon plant wounding. The research objective is to define the VirA linker/phenol interaction that would help elucidate the molecular basis underlying A. tumefaciens pathogenesis. We have produced soluble protein of the VirA linker region at a purity of ~80% and yield of 10mg/mL for crystallization.
Sickle cell disease is the most common inherited hematologic disorder, affecting about 100,000 people in the US, and over 20 million worldwide. A promising therapy is to increase the non-polymer forming oxygenated HbS concentration with aromatic aldehydes. Several aromatic aldehydes alone and/or derivatized with NO-releasing moieties have been studied for their potential treatment of SCD by Dr. Safo’s group. In this study, we used X-ray crystallography to determine the binding modes of aromatic aldehydes with Hb. The crystallographic studies show the compounds to interact with Hb at the α-cleft of Hb, leading to stabilization of the non-polymer forming R-state HbS, explaining the biological activities of the compounds.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-12-2023