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Title: Structural and mechanistic studies on the biosynthesis of the 3'-deoxy nucleoside of the pacidamycins
Author: Michailidou, Freideriki
ISNI:       0000 0004 7234 3926
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 2018
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Nucleic acids are ubiquitous in nature and modified nucleosides are present in a wide range of anti-viral, anti-cancer drugs and antibiotics. Although a variety of naturally occurring nucleoside analogues exist, few include modifications to the ribose or deoxyribose ring. Intriguingly, the uridyl peptide antibiotics (UPAs), such as pacidamycin, contain a biosynthetically unique 3'-deoxyuridine which resembles synthetic anti-retrovirals. Elucidation of the biosynthesis of this structuraly unique nucleoside motif suggests a degree of substrate flexibility, making it a highly attractive prospect for biosynthetic approaches to nucleoside modification. In order to fully exploit the biotransformative potential, a detailed mechanistic understanding of the individual enzymes involved in the biosynthesis of the nucleoside moiety, and especially the enzyme employed at the installation of the 3'-deoxy modification, is required. Chapter 1, the introduction the thesis, discusses the importance of nucleosides for Chemistry and Biology. The section describes the biosynthesis of the nucleoside antibiotics and reviews the recent advances relating to the synthesis and biosynthesis of 3'-deoxy-nucleosides. The Chapter proceeds to describes the biosynthesis of deoxy-sugars, deoxy-nucleosides and nucleotides, reviewing the most common dehydratase mechanisms in addition to examining unusual dehydratases involved in carbohydrate metabolism. Chapter 2, the study of Pac13, the uridine-5'-aldehyde dehydratase of the pacidamyicin nucleoside cluster, is reported. Through detailed functional, structural and kinetic analysis of the wild-type enzyme as well a series of mutants, Chapter 2 provides insight into the mechanism emplyed by this unusual enzyme. Chapter 3 describes the structural and functional analysis of Pac11, the flavin-dependent oxidoreductase of the nucleoside biosynthetic cluster, while Chapter 4 revolves around Pac5, the PLP-dependent aminotransferase. In Chapter 5, the chemical synthesis of fluorinated nucleosides, as probes for exploring the enzymes' mechanism is investigated. Chapter 7 reports the experimental procedures for the research described in this document. The work described in this thesis broadens the understanding of the biosynthesis of deoxy-nucleosides and constitutes the first structural and mechanistic study of the biosynthesis of the biosynthesis of the valuable yet, synthetically challenging 3'-deoxy nucleosides.
Supervisor: Goss, Rebecca J. M. Sponsor: Engineering and Physical Sciences Research Council (EPSRC) ; GlaxoSmithKline
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP625.N88M5 ; Nucleosides--Synthesis ; Biosynthesis ; Peptide antibiotics