Post-translational modifications of methyl-coenzyme m reductase in methanosarcina acetivorans
Rodriguez Carrero, Roy Joel
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https://hdl.handle.net/2142/127363
Description
Title
Post-translational modifications of methyl-coenzyme m reductase in methanosarcina acetivorans
Author(s)
Rodriguez Carrero, Roy Joel
Issue Date
2024-11-25
Director of Research (if dissertation) or Advisor (if thesis)
Metcalf, William W
Doctoral Committee Chair(s)
Metcalf, William W
Committee Member(s)
Olsen, Gary J
Cronan, John E
Nair, Satish K
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Methyl-coenzyme M reductase
methanogenesis
post-translational modification
Archaea
Methanosarcina
Abstract
Methyl-coenzyme M reductase (MCR) catalyzes the last step in methanogenesis and the first step in anaerobic methane oxidation, producing gigatons of methane annually and playing a vital role in the biological methane cycle. This enzyme is found solely in methanogenic and methanotrophic archaea, which are ubiquitous microorganisms inhabiting a variety of anoxic environments. Additionally, MCR contains several unusual post-translationally modified (PTM) amino acids near its active site. Although some of these modifications are widely conserved, others are rarely observed. However, all MCRs characterized to date have at least three post-translationally modified amino acids, suggesting an important role in either the catalysis or the structure of the enzyme’s active site. Although the genes responsible for some of the modifications have been elucidated, the identity of the genes required for the modification of the remaining modified amino acids remain unknown. One of these modifications, 2-methylglutamine was shown to be synthesized by a B12-dependent radical SAM (rSAM) enzyme, designated MgmA. In Chapter 2, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans, an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. In Chapter 3, we determined the MCR modification profiles from Methanobacterium formicicum, Methanomassiliicoccus luminyensis and Methanospirillum hungatei. Previously, the genes required for the PTMs found in MCR have been found using a combination of comparative genomics, genomic context, and gene homology. A limiting factor in the effort of identifying these genes through bottom-up genetic approaches is the low number of MCRs with known modification profiles. Tandem MS and electron density analysis of the MCR from M. formicicum revealed that it lacks 1(N)-methylhistidine and thioglycine, two conserved post translational modifications. Additionally, another pattern observed in both M. formicicum and M. luminyensis was the absence of thioglycine despite the presence of homologs of ycaO and tfuA, the genes required for the synthesis of thioglycine. Finally, in Chapter 4 I summarize all my findings throughout the previous chapters and outline future endeavors that will help in the effort of discovering the identity of the genes required for the remaining PTMs in MCR, as well as in unraveling the effect that these PTMs have in its activity.
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