A Story About Light and Energy: Mechanistic and Biosynthetic Investigations of Bioluminescence and Methanogenesis
Type of DegreePhD Dissertation
Chemistry and Biochemistry
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This dissertation seeks to investigate underexplored aspects of dinoflagellate bioluminescence and biological methane production. Ubiquitous in the four oceans, dinoflagellate bioluminescence is a consequence of an oxidation of a luciferin (LH2) substrate by the luciferase (LCF) enzyme. Utilizing an array of analytical techniques including high-performance liquid chromatography (HPLC), mass spectrometry (MS), and UV-visible/fluorescence spectroscopy, this work initially probes the biosynthesis of dinoflagellate LH2. Pyropheophorbide a is incubated with cell free extracts of Pyrocystis fusiformis, the dinoflagellate model organism of this study. Analysis of the reaction mixtures revealed two novel chlorophyll catabolites, P710 and P680, which we propose are involved in the dinoflagellate LH2 biosynthetic pathway. Additionally, the structure of P630, the previously discovered immediate precursor of dinoflagellate LH2, was assigned. Computational methods, including constant pH accelerated molecular dynamics (CpHaMD) and time-dependent long-range corrected density functional theory (TDLCDFT), were used to probe the pH regulation of dinoflagellate LCF and the mechanism of the bioluminescence reaction, respectively. CpHaMD simulations carried out on domain III of dinoflagellate LCF demonstrated that at pH 8, the pH at which LCF is maintained in a physiologically inactive state, the conformation of domain III is relative stable, exhibiting little to no large-scale conformational fluctuations. In contrast, an identical simulation carried out on domain III of LCF at pH 6, which is close to the pH optimum of LCF, displayed large collective movements of the proposed regulatory α-helical bundle of the N-terminal region. We also carried out TDLCDFT calculations on proposed excited state (peroxy anion, hydroperoxide, gem-diol, and gem-diolate) intermediates of the LH2-LCF reaction mechanism in an effort to identify the bioluminophore (the light emitting species). Contrary to previously proposed reaction mechanisms, analysis of the first ten low-lying excited states revealed that the most energetically feasible and consistent excited state transition energy resided with the gem-diolate intermediate undergoing a biologically novel twisted intermolecular charge transfer mechanism. Biological methane production is carried out by microorganisms classified as methanogenic archaea. Arguably the most significant reaction in methanogenesis, the terminal step catalyzed by the enzyme methyl-coenzyme M reductase (MCR), uses methyl-coenzyme M and coenzyme B as substrates, coenzyme F430 as a cofactor, and produces methane gas. In an effort to construct active holo MCR in a heterologous host, the possibility of in vivo biosynthesis of F430 in Escherichia coli was investigated. Coexpression of all previously established coenzyme F430 genes and HPLC/MS analysis of the cell extracts indicated that F430 was not synthesized with the coenzyme F430 genes alone, with the pathway ending prematurely at Ni-sirohydorchlorin a,c-diamide. Additionally, to elucidate the origin and purpose of several post-translational modifications (PTMs) discovered within the active site region of MCR, a unique cloning/expression strategy was developed and complemented with MS. Genes from the mcr gene cluster, mcrGBDC, were ligated into a single plasmid with each gene possessing its own promoter and ribosome-binding site. The mcrA gene, equipped with a C-terminal hexahistidine tag for protein purification purposes, was coexpressed on a separate plasmid with the mcrGBDC genes and plasmids containing genes proposed to be involved in performing the PTMs. MS analysis of purified McrA revealed that a protein methylation gene A (prmA) homolog was responsible for the 1-N-methylhistidine and S-methylcysteine modifications. Additionally, gene knockout experiments demonstrated that methanogenesis marker 10 (mm10) is responsible for the 5-(S)-methylarginine PTM.