Author ORCID Identifier

https://orcid.org/0009-0001-1229-8516

Date Available

3-16-2024

Year of Publication

2023

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

Antibiotic resistance happens when bacteria develop the ability to survive medications that normally terminate them. Instead, these super germs are able to survive in the body and produce a community of antibiotic resistance germs which can cause human fatalities. It is important to discover and develop new compounds and molecules that will improve this clinical obstacle. This research focused on analyzing the biosynthesis that incorporates distinctive chemical characteristic of various nucleoside antibiotics, ß-hydroxy amino acids and α-methyl-amino acids. ß-hydroxy amino acids and α-methyl-amino acids are considered an important class of industrially useful compounds, particularly for pharmaceutical development, and are found in a variety of natural products. A solution to antibiotic resistance is to use enzyme catalysis as an biocatalytic tool to build nucleoside antibiotics that target overlooked drug inhibition. One study will focus on investigating the enzymatic synthesis of Muraymycin which targets MraY, a novel target for antibiotic development during the production of the peptidoglycan cell wall. Phospho-MurNAc-pentatpeptide translocase I (MraY) is an essential enzyme for peptidoglycan development. Muraymycin is a natural product that targets MraY and also encompasses a C7 sugar which is named 5’-C-glycyluridine (GlyU). GlyU has the structural feature of a ß-hydroxy-α-serine. The biosynthetic gene cluster for A-90289 contains a gene product (LipK) which has shown the ability to catalyze a PLP-dependent conversion of L-Thr and U5’A to acetaldehyde and GlyU. By mirroring the LipK reaction to another transaldolase enzyme, MpTA deriving from the same homolog, has shown the ability to catalyze the reaction between L-Thr and uridine 5’-aldehyde to form a GlyU precursor by α-replacement chemistry. This shows how transaldolases could be used as a structural diversification tool and has a broad specificity for aldehydes. Amicetin, an aminohexose-cytosine antibiotic with antibacterial and antiviral properties is part of the nucleoside antibiotic family. Nucleoside antibiotics are a family of biologically varied natural product compounds with unique structural characteristics. Amicetin features an α-methyl serine within the C terminus. During this study, Streptomyces vinaceusdrappus NRRL 2363 was cultivated for the Amicetin biosynthesis gene cluster to be cloned. The amicetin biosynthetic gene cluster is heterologously expressed in PM-1 media resulting in the production of amicetin, which confirms the production of the ami gene cluster. Bioinformatic data analysis showed 21 genes involved in the biosynthesis, including 2 aiding in the downstream linkage of p-aminobenzoic acid (PABA) and the terminal α-methyl serine. The activity of these enzymes, amiR and amiT, were tested via activity bioassay to confirm hypothesized functionality. Mutations were designed to cause the inactivation of the nonribosomal peptide synthase (NRPS) adenylation domain gene amiT and the N-acetyltransferase gene amiR which led to unsuccessful linkage of methyl serine moiety to plicacetin. These results exhibit the responsibility of AmiT recognizing α-methyl-L-serine and α-methyl-D-serine and AmiR catalyzing the formation of the amide bond linking PABA and methyl serine during downstream biosynthesis of Amicetin. By using multi-module enzymes as biocatalytic tools, could help combat antibiotic resistance by diversifying current pharmaceuticals available.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.425

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