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 (rMCR) was expressed in the mesophile The rMCR was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F Subunits of the native (MCR) 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 from and the from in The His-tagged purified rMCR then contained the McrA and the 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. 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 F 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.

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][BF] ionic liquid as compared to the hexafluorophosphate-based [BMIM][PF] and conventional organic solvents. However, the origin of the rate difference between [BMIM][BF] and [BMIM][PF] 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 pre-ordered 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][BF] as compared to [BMIM][PF].

Major depression is a life threatening psychiatric disorder. Hyperactivity of HPA axis is one of the pathophysiological changes of depression, and HPA axis is known to be under the neural control of mPFC, hippocampus and amygdala. Besides, FKBP5 is an inhibitory factor of glucocorticoid receptor (GR), and single nucleotide polymorphisms (SNPs) of FKBP5 is reported to be significantly correlated with depression. Here we investigated the effect of single and repeated restraint stress (RS) (3 hr/day) on depression-and anxiety-like behaviors using forced swimming test (FST) and elevated plus maze test (EPM) in rats. Moreover, we assessed the effect of repeated RS on the serum corticosterone by EIA, and on the GR and FKBP5 in the mPFC, hippocampus and amygdala by Western blotting. We performed above these assessments 2 weeks after repeated stress. Seven times RS increased depression-like behavior (immobility in FST) (**P< 0.017), and it was antagonized by previous 14 days repeated administration of escitalopram (10 mg/kg), a SSRI (*P< 0.0083). Locomotor activity and anxiety assessed by EPM were no change. Serum corticosterone was increased by seven times RS (**P< 0.017). Protein level of GR was not changed, but that of FKBP5 was increased in the amygdala by seven times RS (**P< 0.05). Hyperactivity of HPA axis and increased FKBP5 in the amygdala may be related to RS-induced depression-like behavior.