Author ORCID Identifier

https://orcid.org/0000-0003-2280-2461

Date Available

12-2-2023

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. Steven G. Van Lanen

Abstract

New antibiotic scaffolds with novel drug targets are needed to combat the rise of drug-resistant, infectious microorganisms. The bacterial translocase I is a ubiquitous enzyme in the peptidoglycan biosynthetic pathway that has yet to be targeted by clinically used antibiotics. It catalyzes the transfer of N-acetylmuramoyl-pentapeptide to undecaprenylphosphate in order to generate lipid I during cell wall biosynthesis. A screening of bacterial translocase I inhibitors led to the discovery of the novel compound capuramycin and its analogues: A-500359s, A-503083s, and A-102395, produced by various species of actinomycetes. The capuramycins show potent activity against the bacterial translocase I with IC50s in the nanomolar range, and semi-synthetic analogues have been shown to eradicate both active and dormant forms of Mycobacterium tuberculosis.

Recently, a new analogue of capuramycin with comparable antibiotic activity has been discovered. This compound, named capuramycin B, differs from other known capuramycins in that it contains a unique propanamide group that extends from the characteristic 5′-carboxamide. The genome of the producing strain for this new compound has been sequenced, revealing a biosynthetic gene cluster of approximately 54-kb DNA. The organization and homology of the gene cluster most closely resembles those of the A-500359 and A-503083 gene clusters. Interestingly, the gene encoding a self-resistance phosphotransferase (capP for A-503083 biosynthesis) is prematurely truncated in the capuramycin B gene cluster. Additionally, three open reading frames are present at the end of the gene cluster that differ significantly from the other gene clusters in homology and putative function. One is a putative, distinct phosphotransferase, CpuV; one is a putative nonribosomal peptide synthetase, CpuW; and one is a putative Bac luciferase, CpuY.

In this study, we aim to demonstrate the activities of the gene products of these three new orfs. First, we functionally assign CpuV as a capuramycin B phosphotransferase that imparts self-resistance to capuramycins. Second, we propose the ability of CpuW and CpuY to modify an amino acid to generate a potential precursor to the unique propanamide group. The ultimate goal of this work is to understand the full biosynthetic mechanism of capuramycin-type antibiotics for the rational design therapeutically valuable analogs.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.408

Funding Information

This study was supported by the National Institutes of Health National Institute of Allergy and Infectious Diseases Grants (no.: AI087849 and no.: AI128862) in 2016.

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