Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

Metalloproteins requiring one or more metal ions for normal function make up 30% of all known proteins, and many critical biological pathways contain at least one metallo-enzyme. Di-nuclear metallo-proteins constitute a large class of these proteins yet we currently lack effective methods of inhibiting these enzymes for the development of new medical therapies, particularly for the discovery of new antibiotics. Our work has focused on developing novel functionalities that selectively interact with di-nuclear catalytic centers, and we are targeting three separate di-zinc-metallo-enzymes that are unique to bacteria and play key roles in their growth and development. These enzymes are DapE, AiiA, and NDM-1. DapE is involved in biosynthesis of lysine and meso-diaminopimelic acid, essential precursors in the production of bacterial cell walls. AiiA is a di-Zn-dependent lactonase involved in bacterial cell-cell communication, and NDM-1 is a di-metallo-beta-lactamase capable of deactivating the most commonly administered antibiotics, gaining international attention for this enzyme as a clinically-relevant pharmaceutical target, yet drug development efforts have proven ineffective due to a lack of effective inhibitors.

As part of our ongoing studies to functionally annotate the Gcn5-related N-acetyltransferase (GNAT) PA4794 from Pseudomonas aeruginosa with unknown functions, we have used PA4794 as a model system for exploring efficient formation of bisubstrate complexes to enhance our success rate in obtaining co-crystal structures of GNATs with ligands bound in their acceptor sites. We have synthesized and tested substrate analogs of the previously identified N-phenylacetyl glycine lysine (NPAcGK) enabling two separate three-dimensional structures of PA4794 with NPAcGK analog-derived bisubstrates formed through direct reaction with CoA—the first through direct alkylation with a reactive substrate, and the second through X-ray induced radical-mediated process. We have also performed docking and molecular dynamics simulations of the reverse reaction pathway from the NPAcGK product back to formation of the tetrahedral intermediate/transition state to complement our structural work and to explore the key ligand-protein interactions within the active site of PA4794, guiding mutant synthesis and kinetics to explore the role of key residues in the active site.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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