Assembly of Methyl Coenzyme M Reductase in the Methanogenic Archaeon Methanococcus maripaludis.

Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile Methanothermococcus okinawensis (rMCRok) was expressed in the mesophile Methanococcus maripaludis The rMCRok was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F430 Subunits of the native M. maripaludis (MCRmar) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-tagged mcrA from M. maripaludis and the mcrBDCG from M. okinawensis in M. maripaludis The His-tagged purified rMCR then contained the M. maripaludis McrA and the M. okinawensis McrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.IMPORTANCE Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F430 cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.

Examining Ionic Liquid Effects on Mononuclear Rearrangement of Heterocycles Using QM/MM Simulations.

The mononuclear rearrangement of heterocycles (MRH) reaction of the Z-phenylhydrazone of 3-benzoyl-5-phenyl-1,2,4-oxadiazole into 4-benzoylamino-2,5-diphenyl-1,2,3-triazole derives a sizable rate enhancement in the 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4] ionic liquid as compared to the hexafluorophosphate-based [BMIM][PF6] and conventional organic solvents. However, the origin of the rate difference between [BMIM][BF4] and [BMIM][PF6] has proven difficult to rationalize as no experimental trend relates the physical properties of the solvents, e.g., polarity and viscosity, to the rates of reaction. QM/MM calculations in combination with free-energy perturbation theory and Monte Carlo sampling have been carried out for the MRH reaction to elucidate the disparities in rates when using ionic liquids, methanol, and acetonitrile. Activation barriers and solute-solvent interactions have been computed for both an uncatalyzed and a specific base-catalyzed mechanism. Energetic and structural analyses determined that favorable π+-π interactions between the BMIM cation, the substrate phenyl rings, and the bicyclic quasi-aromatic 10π oxadiazole/triazole transition state region imposed a preordered geometric arrangement that enhanced the rate of reaction. An ionic liquid clathrate formation enforced a coplanar orientation of the phenyl rings that maximized the electronic effects exerted on the reaction route. In addition, site-specific electrostatic stabilization between the ions and the MRH substrate was more prevalent in [BMIM][BF4] as compared to [BMIM][PF6].