scholarly journals Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A

2018 ◽  
pp. 325-347 ◽  
Author(s):  
Dipti D. Nayak ◽  
William W. Metcalf
Keyword(s):  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Genetic Techniques ◽  
Methyl Coenzyme M Reductase

scholarly journals Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

2017 ◽  
Author(s):  
Dipti D. Nayak ◽  
Nilkamal Mahanta ◽  
Douglas A. Mitchell ◽  
William W. Metcalf
Keyword(s):  
Strictly Anaerobic ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Α Subunit ◽  
Post Translational Modifications ◽  
Physiological Characterization ◽  
Key Enzyme ◽  
Catalytic Role ◽  
Methyl Coenzyme M Reductase

AbstractThe enzyme methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α subunit of this enzyme (McrA) contains several unusual post-translational modifications, including an exceptionally rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioamide-containing natural products, we hypothesized that the archaealtfuAandycaOgenes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA in a ΔycaO-tfuAmutant of the methanogenic archaeonMethanosarcina acetivoransrevealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Physiological characterization of this mutant suggested a new role for the thioglycine modification in enhancing protein stability, as opposed to playing a direct catalytic role. The universal conservation of this modification suggests that MCR arose in a thermophilic ancestor.

scholarly journals Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Dipti D Nayak ◽  
Nilkamal Mahanta ◽  
Douglas A Mitchell ◽  
William W Metcalf
Keyword(s):  
Elevated Temperatures ◽  
Protein Secondary Structure ◽  
Phenotypic Characterization ◽  
Strictly Anaerobic ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Α Subunit ◽  
Key Enzyme ◽  
Methyl Coenzyme M Reductase

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.

scholarly journals Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

2019 ◽  
Author(s):  
Dipti D Nayak ◽  
Andi Liu ◽  
Neha Agrawal ◽  
Roy Rodriguez-Carerro ◽  
Shi-Hui Dong ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methanogenic Archaea ◽  
Physical Structure ◽  
Major Influence ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Phenotypic Analysis ◽  
And Function ◽  
Methyl Coenzyme M Reductase

AbstractMethyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six post-translationally modified amino acids near the active site. Recent studies have identified genes that install two of these modifications (thioglycine and 5-(S)-methylarginine), yet little is known about the installation and function of the remaining post-translationally modified residues. Here, we provide in vivo evidence that a dedicated SAM-dependent methyltransferase encoded by a gene we designated mcmA is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue plays an important role in adaptation to a mesophilic lifestyle. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. Surprisingly, high-resolution crystal structures of the various unmodified MCRs were indistinguishable from the fully modified enzyme, suggesting that interactions between the post-translationally modified residues do not exert a major influence on the physical structure of the enzyme, but rather serve to fine-tune the activity and efficiency of MCR.

scholarly journals Methanol-Dependent Gene Expression Demonstrates that Methyl-Coenzyme M Reductase Is Essential in Methanosarcina acetivorans C2A and Allows Isolation of Mutants with Defects in Regulation of the Methanol Utilization Pathway

2005 ◽  
Vol 187 (16) ◽  
pp. 5552-5559 ◽  
Author(s):  
Michael Rother ◽  
Paolo Boccazzi ◽  
Arpita Bose ◽  
Matthew A. Pritchett ◽  
W. W. Metcalf
Keyword(s):  
Proteome Analysis ◽  
Genetic System ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Prolonged Incubation ◽  
Methanol Utilization ◽  
Expression Of Genes ◽  
Regulatory Mutations ◽  
Utilization Pathway ◽  
Methyl Coenzyme M Reductase

ABSTRACT Methanosarcina acetivorans C2A is able to convert several substrates to methane via at least four distinct methanogenic pathways. A common step in each of these pathways is the reduction of methyl-coenzyme M (CoM) to methane catalyzed by methyl-CoM reductase (MCR). Because this enzyme is used in each of the known pathways, the mcrBDCGA operon, which encodes MCR, is expected to be essential. To validate this prediction, a system for conditional gene inactivation was developed. A heterologous copy of the mcrBDCGA operon was placed under the control of the highly regulated mtaC1 promoter, which directs the expression of genes involved in methanol utilization, and recombined onto the M. acetivorans chromosome. This allowed for disruption of the endogenous mcr operon in the presence of methanol. Because the PmtaC1 promoter is transcribed only during growth on methanol, mcrBDCGA was rendered methanol dependent and the strain was unable to grow in trimethylamine media, strongly suggesting that mcrBDCGA is essential. Upon prolonged incubation, suppressed mutants which expressed mcrBDCGA constitutively could be selected. Expression analysis of PmtaC1::uidA gene fusions in several isolated suppressed mutants suggests that they carry trans-active mutations leading to deregulation of all genes under control of this promoter. Subsequently, proteome analysis of one such suppressed mutant revealed that all known proteins derived from mtaC1 promoter-dependent expression were constitutively expressed in this mutant. This genetic system can therefore be employed for the testing of essential genes and for the identification of genes under a common regulatory mechanism by making regulatory mutations phenotypically selectable.

scholarly journals Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

PLoS Biology ◽  
2020 ◽  
Vol 18 (2) ◽  
pp. e3000507 ◽  
Author(s):  
Dipti D. Nayak ◽  
Andi Liu ◽  
Neha Agrawal ◽  
Roy Rodriguez-Carerro ◽  
Shi-Hui Dong ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Functional Interactions ◽  
Methyl Coenzyme M Reductase

scholarly journals Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-6
Author(s):  
Yanli Zhang ◽  
Linley R. Schofield ◽  
Carrie Sang ◽  
Debjit Dey ◽  
Ron S. Ronimus
Keyword(s):  
Biosynthetic Pathway ◽  
Critical Role ◽  
Reduction Reaction ◽  
Coenzyme M ◽  
Purification And Characterization ◽  
The Third ◽  
Optimal Activity ◽  
Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

scholarly journals Anti-Methanogenic Effect of Phytochemicals on Methyl-Coenzyme M Reductase—Potential: In Silico and Molecular Docking Studies for Environmental Protection

Micromachines ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1425
Author(s):  
Yuvaraj Dinakarkumar ◽  
Jothi Ramalingam Rajabathar ◽  
Selvaraj Arokiyaraj ◽  
Iyyappan Jeyaraj ◽  
Sai Ramesh Anjaneyulu ◽  
...  
Keyword(s):  
Molecular Docking ◽  
In Silico ◽  
Rosmarinic Acid ◽  
Docking Studies ◽  
Thymus Vulgaris ◽  
Piper Betle ◽  
Coenzyme M ◽  
Dynamics Simulations ◽  
Autodock 4.0 ◽  
Methyl Coenzyme M Reductase

Methane is a greenhouse gas which poses a great threat to life on earth as its emissions directly contribute to global warming and methane has a 28-fold higher warming potential over that of carbon dioxide. Ruminants have been identified as a major source of methane emission as a result of methanogenesis by their respective gut microbiomes. Various plants produce highly bioactive compounds which can be investigated to find a potential inhibitor of methyl-coenzyme M reductase (the target protein for methanogenesis). To speed up the process and to limit the use of laboratory resources, the present study uses an in-silico molecular docking approach to explore the anti-methanogenic properties of phytochemicals from Cymbopogon citratus, Origanum vulgare, Lavandula officinalis, Cinnamomum zeylanicum, Piper betle, Cuminum cyminum, Ocimum gratissimum, Salvia sclarea, Allium sativum, Rosmarinus officinalis and Thymus vulgaris. A total of 168 compounds from 11 plants were virtually screened. Finally, 25 scrutinized compounds were evaluated against methyl-coenzyme M reductase (MCR) protein using the AutoDock 4.0 program. In conclusion, the study identified 21 out of 25 compounds against inhibition of the MCR protein. Particularly, five compounds: rosmarinic acid (−10.71 kcal/mol), biotin (−9.38 kcal/mol), α-cadinol (−8.16 kcal/mol), (3R,3aS,6R,6aR)-3-(2H-1,3-benzodioxol-4-yl)-6-(2H-1,3-benzodioxol-5-yl)-hexahydrofuro[3,4-c]furan-1-one (−12.21 kcal/mol), and 2,4,7,9-tetramethyl-5decyn4,7diol (−9.02 kcal/mol) showed higher binding energy towards the MCR protein. In turn, these compounds have potential utility as rumen methanogenic inhibitors in the proposed methane inhibitor program. Ultimately, molecular dynamics simulations of rosmarinic acid and (3R,3aS,6R,6aR) -3-(2H-1,3-benzodioxol-4-yl)-6-(2H-1,3-benzodioxol-5-yl)-hexahydrofuro[3,4-c]furan-1-one yielded the best possible interaction and stability with the active site of 5A8K protein for 20 ns.

Comparison of chemical properties of iron, cobalt, and nickel porphyrins, corrins, and hydrocorphins

2005 ◽  
Vol 09 (08) ◽  
pp. 581-606 ◽  
Author(s):  
Kasper P. Jensen ◽  
Ulf Ryde
Keyword(s):  
Density Functional ◽  
Hydrolysis Product ◽  
Chemical Properties ◽  
Coenzyme M ◽  
Ring Systems ◽  
Reduction Potentials ◽  
Higher Spin ◽  
Coenzyme F430 ◽  
Coenzyme B ◽  
Methyl Coenzyme M Reductase

Density functional calculations have been used to compare the geometric, electronic, and functional properties of the three important tetrapyrrole systems in biology, heme, coenzyme B 12, and coenzyme F430, formed from iron porphyrin ( Por ), cobalt corrin ( Cor ), and nickel hydrocorphin ( Hcor ). The results show that the flexibility of the ring systems follows the trend Hcor > Cor > Por and that the size of the central cavity follows the trend Cor < Por < Hcor . Therefore, low-spin Co I, Co II, and Co III fit well into the Cor ring, whereas Por seems to be more ideal for the higher spin states of iron, and the cavity in Hcor is tailored for the larger Ni ion, especially in the high-spin Ni II state. This is confirmed by the thermodynamic stabilities of the various combinations of metals and ring systems. Reduction potentials indicate that the +I and +III states are less stable for Ni than for the other metal ions. Moreover, Ni – C bonds are appreciably less stable than Co - C bonds. However, it is still possible that a Ni – CH 3 bond is formed in F 430 by a heterolytic methyl transfer reaction, provided that the donor is appropriate, e.g. if coenzyme M is protonated. This can be facilitated by the adjacent SO 3− group in this coenzyme and by the axial glutamine ligand, which stabilizes the Ni III state. Our results also show that a Ni III– CH 3 complex is readily hydrolysed to form a methane molecule and that the Ni III hydrolysis product can oxidize coenzyme B and M to a heterodisulphide in the reaction mechanism of methyl coenzyme M reductase.

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