Methyl-coenzyme M reductase (MCR) is the key enzyme involved in the production of methane by methanogenic archaea and its consumption by anaerobic methanotrophs (ANME). MCR is a multimeric complex composed of six different subunits arranged in a 2α, 2β, 2γ configuration that requires two molecules of its nickel-containing tetrapyrrole prosthetic group, coenzyme F430. Additionally, the α subunits of MCR house a variety of different post-translational modifications across both methanogens and ANME. In methanogens, MCR is encoded in a conserved mcrBDCGA gene cluster, which encodes accessory proteins McrD and McrC. These are believed to be involved in the assembly and activation of MCR, respectively. However, one or both accessory proteins are often omitted from the operon in other MCR-containing archaea as is the case in ANME.
MCR knowledge is mostly limited to methanogens due to difficulties associated with large-scale cultivation of ANME and other MCR-containing archaea. Due to the complexity of MCR, studies on this enzyme are also largely limited to native enzymes. Developing methods for the detailed biochemical characterization ANME MCRs would be highly desirable since these enzymes are proposed to be optimized for methane oxidation and thus have immense potential for bioenergy and greenhouse gas mitigation applications. In addition to containing the necessary machinery for the production of an assembled and active MCR, model methanogens are easier to culture and have established genetic manipulation techniques, making them ideal candidates for the development of heterologous expression systems. Thus, here we sought to generate such a system for the study of various ANME MCRs in the methanogen, Methanococcus maripaludis. We report the successful expression and purification of an ANME-2d MCR, marking a significant step toward the development of a heterologous MCR expression system. Additionally, our attempts to purify various recombinant MCRs revealed the importance of including accessory proteins, particularly McrD, within expression constructs. Therefore, we also sought to functionally characterize McrD, which we show is likely an MCR chaperone that plays a key role in MCR maturation. Taken together, our work has provided key insights into MCR assembly as well as provided a foundation for the eventual development of MCR based biocatalytic systems to be used for methane mitigation strategies and bioenergy platforms. / Doctor of Philosophy / Life is divided into three domains known as Bacteria, Eukarya, and Archaea. Methanogens are anerobic microbes belonging to the domain Archaea, which can be found across a wide variety of oxygen deprived environments. These organisms can turn different carbon-containing compounds into energy and methane gas in a process known as methanogenesis. This results in roughly 90 billion tons of biologically produced methane, making methanogenesis a key point of interest for potential greenhouse gas mitigation. The methane-generating step of methanogenesis is performed by methyl-coenzyme M reductase (MCR), a large enzyme composed of two α subunits, two β subunits, and two γ subunits. Additionally, this enzyme harbors a nickel-containing cofactor which is responsible for catalyzing the difficult methane formation reaction. In addition to the MCR-encoding genes, MCR gene clusters contain two extra genes that encode accessory proteins, named McrC and McrD, which are believed to play an important role in the activation and the assembly of the enzyme, respectively.
Relatives of methanogens known as Anerobic Methanotrophs (ANME) are a different type of archaea which consume methane by reversing methanogenesis in a process known as anerobic methane oxidation. Because of their ability to consume methane, there is a large interest in studying MCR from these organisms to potentially use it for methane mitigation strategies and for bioenergy applications to convert methane to more usable liquid fuels. However, due to the high difficulty of growing ANME in a lab setting, studying any biochemical processes from ANME is a difficult task. Luckily, genetic manipulation techniques are available for many methanogens, making them ideal candidates to study MCR from ANME organisms. In this work, we sought to develop a system to express and purify MCR from different methanogens and ANME in a methanogenic host, Methanococcus maripaludis. We also sought to understand the role and importance of accessory protein McrD, especially with respect to developing a proper expression system for MCRs. We were able to successfully express a ANME MCR in M. maripaludis and found that McrD is an important aspect to consider when expressing MCRs in a methanogen, although it is not essential for this protein to exist within the MCR gene cluster. This work sets the stage for the future biotechnological use of MCR for methane mitigation and bioenergy applications.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/113412 |
| Date | 24 January 2023 |
| Creators | Gendron, Aleksei |
| Contributors | Biochemistry, Allen, Kylie Dawn, Helm, Richard F., Slade, Daniel Joseph, White, Robert H. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | Creative Commons Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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