Abstract

The marked increase of bacteria that are resistant to clinically relevant antibiotics in recent years has sparked a push in the development and discovery of novel antibiotics. Complementary to this, the identification of novel antibiotic targets is also of great interest. The lysine biosynthesis pathway is an ideal candidate for these efforts because its product is an essential amino acid. Lysine cannot be synthesized by humans and thus poses minimal risk of side effects if synthesis is targeted. The penultimate product in the lysine biosynthesis pathway, meso-DAP, is also a key component of the gram-negative bacterial cell wall peptidoglycan layer. The L,L-diaminopimelate aminotransferase (DapL) pathway, a recently discovered variant in the lysine biosynthetic pathway is especially of interest because it has been identified in only about 13% of bacteria. Because of its narrow distribution in bacteria, it could be an ideal target for the development of narrow spectrum antibiotics. A key enzyme in the pathway, DapL is a homodimer that catalyzes the conversion of tetrahydrodipicolinate (THDP) to L,L-diaminopimelate (L,L-DAP) in a single, reversible transamination reaction. While this enzyme is essential in plants that contain the pathway, it is not directly known whether the same is true for bacteria that contain the pathway. In order to evaluate DapL as a target for the development of narrow spectrum antibiotics, genetic and phenotypic characterizations must be performed. This project evaluated DapL from Verrucomicrobium spinosum, a close relative of Chlamydia, as a potential target in the development of antibiotic compounds. It assessed the essentiality of the VsdapL gene via targeted knockouts using the CRISPR/cas9 system, providing evidence supporting the essentiality of the gene in bacteria. It also evaluated putative antagonistic ligands using a comprehensive and comparative molecular dynamics (MD) software package – DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0, providing insight into the dynamic behavior of the DapL protein and identifying a correlation between total effect on protein dynamics in silico and inhibition measured in vitro.

Library of Congress Subject Headings

Aminotransferases--Analysis; Enzymes--Analysis; Molecular dynamics; Lysine--Synthesis; Peptidoglycans--Synthesis

Publication Date

2019

Document Type

Thesis

Student Type

Graduate

Degree Name

Bioinformatics (MS)

Department, Program, or Center

Thomas H. Gosnell School of Life Sciences (COS)

Advisor

Andre O. Hudson

Advisor/Committee Member

Gregory A. Babbitt

Advisor/Committee Member

Michael A. Savka

Campus

RIT – Main Campus

Plan Codes

BIOINFO-MS

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