The Modification and Role of a Methyl-Arginine Post-Translational Modification in the Active Site of the Enzyme Methyl-Coenzyme M Reductase
Type of DegreePhD Dissertation
Chemistry and Biochemistry
Restriction TypeAuburn University Users
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The enzyme methyl-coenzyme M reductase (Mcr) is used in methanogenic archaea to produce methane and in anaerobic methanotrophs to oxidize methane. A significant portion of the methane produced by methanogens is released to the atmosphere, where it contributes to global warming. Understanding Mcr could lead to the inhibition of methane production or the optimization of the enzyme to start the conversion of methane into biofuels. Chapter 1 provides a background to the work that will be discussed in this dissertation. Chapter 2 examines methanogenesis marker protein 10 (Mmp10), a proposed radical SAM enzyme that may modify an arginine residue within Mcr. Multiple in vivo studies have examined how knocking out Mmp10 affects whole cells. Here, in vitro experiments with Mmp10 are used to study its methylating ability and investigate the cofactors present in this enzyme. Mass spectrometry show that Mmp10 can methylate the target arginine within a substrate peptide modeled after Mcr. Spectroscopy reveals that Mmp10 contains both a [4Fe-4S](II)/(I) cluster and a cobamide cofactor. In Chapter 3, the activation of Mcr in Methanococcus maripaludis is studied. Mcr is only active in the Ni(I) (Mcr-red1) form, but purifying Mcr without pretreatment produces only the Ni(II) (Mcr-silent) form. Purified Mcrsilent cannot be converted chemically to Mcrred1; however, the Ni(III) (Mcr-ox1) form of Mcr can be reduced to Mcr-red1 in vitro. These experiments examine how the Mcr-ox1 or Mcr-red1 forms can be induced in the whole cells through incubation with sodium sulfide or 100% H2, respectively. As shown by spectroscopy, Mcr-red1 can be induced in whole cells grown on 80% H2/20% CO2 and incubated with 100% H2; however, Mcrox1 cannot be induced when formate-grown cells are incubated with sodium sulfide. Chapter 4 investigates the iron-sulfur cluster content in a radical SAM enzyme, MptM. The primary sequence of MptM reveals two CX3CX2C motifs that could bind SAM clusters and other potential binding sites for [4Fe-4S](II)/(I) clusters. Site-directed mutagenesis was performed on multiple potential cluster sites and the samples were analyzed with electron paramagnetic resonance (EPR) spectroscopy. The data reveals that there are three [4Fe-4S](II)/(I) clusters within MptM.