The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite the current challenges in NP research, these molecules are still relevant today as they are a major source of human medicine as well as being useful biological tools. The elucidation of their biosynthetic pathways has also provided information about the biochemical and biophysical properties of fascinating enzyme families such as the α- oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’- phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several important NP, including those essential for life. This study reports the characterization and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E. coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is responsible for its characteristic yellow colour. It binds L-serine as a natural substrate with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray crystallography at a resolution of 4.98 Å, which shows four molecules in the asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal region where the ACP domain is located in the crystal strucuture also suggests intrinsic flexibility and disorder within that region. With the increasing demand for new anti-infective therapies, investigations of the molecular interactions between NPs and their protein targets are vital in understanding the inhibition or activation properties of these molecules. The cysteine transpeptidases known as sortases produced by Gram positive bacteria have been identified as attractive targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray crystallography. This study reports the first high resolution crystal structure of the H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain. Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow, tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6 μM. The interaction resulted in a covalent adduct with the active site cysteine residue (Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to 250 μM with an efficacy cut-off point at higher concentations. These results indicate that chalcone flavonoids are worth further investigation as potential antibiofilm inhibitors. A renewed interest in plant NPs has also led to a collaborative investigation on the antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh preparations of garlic extracts. This investigation reports the first evidence that allicin and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE) against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin (BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit further investigation as adjuncts to existing antibiotics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:679454 |
| Date | January 2015 |
| Creators | Wallock-Richards, Daynea Juaneckah |
| Contributors | Campopiano, Dominic ; Bradley, Mark |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14188 |
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