Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase
ABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-tra...
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American Society for Microbiology
2025-02-01
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03546-24 |
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| author | Roy J. Rodriguez Carrero Cody T. Lloyd Janhavi Borkar Shounak Nath Liviu M. Mirica Satish Nair Squire J. Booker William Metcalf |
| author_facet | Roy J. Rodriguez Carrero Cody T. Lloyd Janhavi Borkar Shounak Nath Liviu M. Mirica Satish Nair Squire J. Booker William Metcalf |
| author_sort | Roy J. Rodriguez Carrero |
| collection | DOAJ |
| description | ABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, 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. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation.IMPORTANCEMethane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies. |
| format | Article |
| id | doaj-art-e96e87ea89a7467ebe48b61277746537 |
| institution | Kabale University |
| issn | 2150-7511 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj-art-e96e87ea89a7467ebe48b612777465372025-02-05T14:00:48ZengAmerican Society for MicrobiologymBio2150-75112025-02-0116210.1128/mbio.03546-24Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductaseRoy J. Rodriguez Carrero0Cody T. Lloyd1Janhavi Borkar2Shounak Nath3Liviu M. Mirica4Satish Nair5Squire J. Booker6William Metcalf7Department of Microbiology, University of Illinois Urbana-Champaign, Champaign, Illinois, USADepartment of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USADepartment of Biochemistry, University of Illinois Urbana-Champaign, Champaign, Illinois, USADepartment of Chemistry, University of Illinois Urbana-Champaign, Champaign, Illinois, USADepartment of Chemistry, University of Illinois Urbana-Champaign, Champaign, Illinois, USADepartment of Biochemistry, University of Illinois Urbana-Champaign, Champaign, Illinois, USADepartment of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USADepartment of Microbiology, University of Illinois Urbana-Champaign, Champaign, Illinois, USAABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, 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. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation.IMPORTANCEMethane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.https://journals.asm.org/doi/10.1128/mbio.03546-24methyl-coenzyme M reductasemethanogenesispost-translational modificationarchaeaMethanosarcina |
| spellingShingle | Roy J. Rodriguez Carrero Cody T. Lloyd Janhavi Borkar Shounak Nath Liviu M. Mirica Satish Nair Squire J. Booker William Metcalf Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase mBio methyl-coenzyme M reductase methanogenesis post-translational modification archaea Methanosarcina |
| title | Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase |
| title_full | Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase |
| title_fullStr | Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase |
| title_full_unstemmed | Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase |
| title_short | Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase |
| title_sort | genetic and biochemical characterization of a radical sam enzyme required for post translational glutamine methylation of methyl coenzyme m reductase |
| topic | methyl-coenzyme M reductase methanogenesis post-translational modification archaea Methanosarcina |
| url | https://journals.asm.org/doi/10.1128/mbio.03546-24 |
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