The Study of the Activation of Primary Terminal C-H Bonds by Rieske Oxygenase Enzymes and the Identification of a Serine Protease with Anti-nociceptive Activity
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Authors
Ramachandra, Manasa
Date
Type
thesis
Language
eng
Keyword
Rieske oxygenase enzymes , oxidative cyclization , Tambjamines , C-H Bond Activation , Serine Protease , Inflammatory Bowel Disease (IBD) , Faecalibacterium prausnitzii , Nociception , Visceral pain
Alternative Title
Abstract
The first project in this thesis focused on the study of the activity of Rieske oxygenase enzymes, TamC and PtTamC. Tambjamines are methoxy-bipyrrole alkaloids isolated from marine invertebrates and bacteria, and possess several biologically useful properties. The first cyclic analogue, tambjamine MYP1, was isolated from a marine bacterium, Pseudoalteromonas citrea (also proposed to exist in Pseudoalteromonas tunicata). TamC and PtTamC, both Rieske oxygenase enzymes (ROs), are proposed to catalyse the cyclisation of tambjamine YP1, a linear analogue, to tambjamine MYP1. The macrocyclisation in MYP1 would involve a C-C bond formation at a primary carbon atom (terminal methyl) of the alkyl chain in YP1. This cyclisation activity was successfully demonstrated by the heterologous co-expression of TamC or PtTamC along with the identified redox partner enzymes of TamC, PcFdx and PcRed, in Escherichia coli, followed by substrate-feeding assays with synthesised tambjamine YP1 and tambjamine BE-18591 as substrates. These are the first reported C-C bond formations being initiated at the terminal methyl group of an alkyl chain in oxidative cyclisations and therefore, the first successful activation of an unreactive terminal C(sp3)-H bond in an oxidative carbocyclisation reaction catalysed by the RO enzymes, TamC and PtTamC. TamC, PtTamC, and PcRed were also successfully heterologously expressed and purified.
The goal of the second project was the identification of serine proteases with anti-nociceptive activity from Faecalibacterium prausnitzii. Inflammatory bowel disease (IBD) induces hyperexcitability in dorsal root ganglion (DRG) nociceptors contributing to abdominal pain. Proteases modulate nociceptive signals in abdominal pain via activation of cell-surface receptors present on sensory neurons. Recent findings indicate that serine proteases from F. prausnitzii, a gut commensal bacterium, can reduce DRG neuron excitability, thereby suppressing abdominal pain. Genome mining and structure simulation tools led to the heterologous expression and purification of three proteases with potential anti-nociceptive activity. Patch clamp experiments conducted by our collaborators at Queen’s University led to the identification of a single protease with anti-nociceptive activity. The successful identification, expression, and purification of the anti-nociceptive serine protease, Clp-fp, from F. prausnitzii is the first step in the microbial modulation of IBD-related abdominal pain.