|This dissertation seeks to investigate the coenzyme F430 biosynthetic pathway and post-translational modification (PTM) enzymes of methyl-coenzyme M reductase (MCR), which will have applications in natural gas-to-liquid fuel conversion strategies and in the development of inhibitors to help reduce natural greenhouse gas emissions.
Methanogenesis, also named biomethanation, is the metabolic production of methane carried out by archaea called methanogens. MCR plays a significant role in methanogenesis and the anaerobic oxidation of methane (AOM). It catalyzes the terminal step of methanogenesis, resulting in the release of methane. Structural studies show MCR as a heterohexamer, with coenzyme F430, a highly reduced, nickel-chelated tetrapyrrole, at two active sites. However, the enzymology of F430 biosynthesis was unknown. Two compounds, precorrin 2 and sirohydrochlorin, had been proposed as biosynthetic precursors of F430. Molecular differences between these precursors and F430 include nickel chelation, two sites of amidation, the formation of two exocyclic rings, and a four- or six-electron ring reduction, respectively. Based on these differences, a comparative genomics approach was utilized to identify genes related to coenzyme F430 biosynthesis (MA3631, MA3626, MA3627, MA3628, and MA3630 in Methanosarcina acetivorans C2A). These genes were cloned, and the encoded enzymes were expressed. Activity assays were developed to determine the function of each enzyme. Besides in vitro experiments, all coenzyme F430 biosynthesis (cfb) genes were cloned in one vector. Coexpression of the genes and following high-performance liquid chromatography (HPLC) analysis of cell extracts indicate that F430 is unable to be synthesized by the cfb genes alone in the heterologous host Escherichia coli. Additionally, it was found that ferredoxin and ferredoxin reductase (FNR) from spinach could support CfbCD catalysis in vitro and was therefore included in the in vivo F430 biosynthesis studies.
Moreover, there are several PTMs surrounding the MCR active sites. To elucidate the origin of the modifications, an in vivo coexpression approach with mass spectrometry (MS) protein sequencing was utilized. A cell line containing mcrA (or mcrABG or ABGDC) was used for McrA expression. The mcrA gene is equipped with either a C-terminal hexahistidine tag or a small ubiquitin-like modifier (SUMO) tag for ease of protein purification and increased protein solubility. MS data indicate that 1-N-methylhistidine and S-methylcysteine modifications are probably carried out by a protein methylation gene A (prma) homolog. Data also indicate that methanogenesis marker 1 (mm1) possibly catalyzes the biosynthesis of thioglycine modification with TfuA and ThiI.