Figure 1.
Organization of cellular metabolism.
Metabolic reactions in a cell can be catalyzed by A, individual enzymes, or B, multienzyme complexes that channel substrates and/or sequester intermediates in a pathway. Pathways in the cell can be connected in series, C, or in parallel by D, metabolic “routers” that channel electrons and substrates to either of two metabolic pathways.
Figure 2.
Comparison of methanogenesis pathways.
A, Hydrogenotrophic methanogenesis in Methanococcus maripaludis. B, Methylotrophic methanogenesis in Methanosarcina acetivorans. Green ovals: energy-conserving reactions. Red ovals: energy-consuming reaction. Please see text for abbreviations.
Figure 3.
XL-MS identification of a multienzyme complex in Methanosarcina.
A, Detection of HdrD complexes. 2 µg crosslinked cell lysates from controls or strains expressing strep-tagged HdrD1 protein were analyzed by Western blot. Arrows indicate the position of strep-tagged HdrD1 monomer and crosslinked high molecular weight (HMW) complexes. * = degraded HdrD1strep protein. B, HdrD co-purified proteins detected by mass spectrometry. Node sizes, line opacity and line widths are proportional to the average peptide hit score of the protein detected in biological duplicates. Dotted lines denote an average score below 100, solid lines denote an average score of 100 and above. Image created with Cytoscape [36]. C, Putative model of the Hdr:ACDS:Mer complex. During methylotrophic growth, both ACDS and Mer use methyl-H4MPT as a substrate. Black dotted lines = electron flow between active sites. HdrE (blue) or HdrD (red), Mer is a tetramer (orange), and ACDS is composed of 5 subunits in a (α2ε2)4β8(γδ)8 configuration (green) [13], [16], [37].
Table 1.
Plasmids and strains used in this study.
Table 2.
HdrD1 protein:protein interactions detected by Mass Spectrometrya.
Figure 4.
Enzymes used by M. acetivorans.
Current pathway models for growth of M. acetivorans on A, methanol + acetate or B, acetate as carbon and energy sources. Enzymes in red are essential despite no defined purpose in the pathway. Green ovals = energy conserving step. Red ovals = energy-consuming step. Please see text for abbreviations.