Structural and Functional Studies of Enzymes From the Methanofuran and Abyssomicin C Biosynthetic Pathways

  • Carl O Marsh

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

The process of methanogenesis is an as of yet partially characterised metabolic process that converts carbon dioxide into methane and hence has implications for the climate of the planet. Methanofuran is a cofactor in methanogenesis responsible for C1 carbon transfer – a key step in the process, and the cofactor’s biosynthesis is a metabolic process conserved in methanogenic Archaea. There are six enzymes currently affiliated with Methanofuran biosynthesis: MfnA, B, C, D, E, F, which have been studied in the hyperthermophile Methanocaldococcus jannaschii. Here, we look at the methanofuran biosynthesis pathway, and specifically the aldolase MfnB, in detail. The enzymes MfnB, C, D, E, F are cloned, expressed and purified for characterisation using circular dichroism for initial analysis of their thermostable capabilities. The structure and function of MfnB is also assessed as it is a key enzyme in the pathway, responsible for the condensation of glyceralde-3-phosphate (GA-3-P) to form 4-(Hydroxymethyl)-2-furancarboxaldehyde-phosphate which is the active furan ring involved in C1 incorporation. Here, the thermostability of a thermophilic and a mesophilic MfnB, from M. jannaschii and Methanococcus maripaludis respectively, is investigated using circular dichroism and molecular dynamics simulations, and its temperature dependent activity for its canonical D-GA-3-P, as well as L-GA-3-P, assessed by UV-spectroscopy. A study into their amino acid compositions, directed by the differing primary sequence and aided by homology modelling, help to justify changes in substrate binding affinities at their respective optimum temperatures. Additionally, mapped mutations in proximity to active site residues are thought to grant protection from heating effects through constraining key active site residue positions in space. This study aims to further characterise the Methanofuran biosynthesis pathway and explore the biosynthetic enzyme’s potential for industrial application.
Date of Award29 Sept 2020
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorPaul R Race (Supervisor) & James E M Stach (Supervisor)

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