scholarly journals Biochemical characterization of the methylmercaptopropionate:cob(I)alamin methyltransferase fromMethanosarcina acetivorans

2019 ◽  
Author(s):  
He Fu ◽  
Michelle N. Goettge ◽  
William W. Metcalf
Keyword(s):  
Transfer Reaction ◽  
Biochemical Characterization ◽  
Detailed Knowledge ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Metabolic Intermediate ◽  
High Substrate ◽  
Methyl Transfer ◽  
Substrate Concentrations

ABSTRACTMethanogenesis from methylated substrates is initiated by substrate specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, theMethanosarcina acetivoransMtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate(MMPA):Coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, withkcatof 0.315 s-1and apparentKmfor MPA of 12 μM. CoM did not serve as a methyl acceptor, thus a second, unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, could also be demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were co-expressed. Biochemical characterization of this complex was not feasible because the corrinoid cofactor of this complex was in the inactive Co(II) state and could not be reactivated by incubation with strong reductants. The MtsF protein, comprised of both corrinoid and methyltransferase domains, co-purifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCEMMPA is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate methyl-CoM. Therefore, additional enzymes required for methanogenic MMPA catabolism await discovery.

scholarly journals Biochemical Characterization of the Methylmercaptopropionate:Cob(I)alamin Methyltransferase fromMethanosarcina acetivorans

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
He Fu ◽  
Michelle N. Goettge ◽  
William W. Metcalf
Keyword(s):  
Transfer Reaction ◽  
Biochemical Characterization ◽  
Detailed Knowledge ◽  
Coenzyme M ◽  
Metabolic Intermediate ◽  
Content Type ◽  
High Substrate ◽  
Methyl Transfer ◽  
Substrate Concentrations

ABSTRACTMethanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, theMethanosarcina acetivoransMtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, withkcatof 0.315 s−1and apparentKmfor MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCEMethylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

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 Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

2013 ◽  
Vol 12 (6) ◽  
pp. 853-863 ◽  
Author(s):  
Karine Lambou ◽  
Andrea Pennati ◽  
Isabel Valsecchi ◽  
Rui Tada ◽  
Stephen Sherman ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Glycine Betaine ◽  
Biochemical Characterization ◽  
Choline Oxidase ◽  
Betaine Aldehyde Dehydrogenase ◽  
Metabolic Intermediate ◽  
Content Type ◽  
Betaine Aldehyde ◽  
Functional Features

ABSTRACTThe choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified inAspergillus fumigatusare present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs.A. fumigatusChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor.A. fumigatusBadhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+to NADH. Analysis of theAfchoAΔ::HPHandAfbadAΔ::HPHsingle mutants and theAfchoAΔAfbadAΔ::HPHdouble mutant showed thatAfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process.AfChoAp andAfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine inA. fumigatusfunctions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study inA. fumigatusis the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

scholarly journals Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism

2020 ◽  
Vol 295 (26) ◽  
pp. 8708-8724
Author(s):  
Van Dung Pham ◽  
Tuan Anh To ◽  
Cynthia Gagné-Thivierge ◽  
Manon Couture ◽  
Patrick Lagüe ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Substrate Inhibition ◽  
Catalytic Triad ◽  
Inhibition Mechanism ◽  
Structural Determinants ◽  
Mutant Proteins ◽  
Carbon And Nitrogen ◽  
Structural Insights ◽  
Substrate Concentrations

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa. Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.

scholarly journals A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhen Yan ◽  
Mingyu Wang ◽  
James G. Ferry
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Biochemical Characterization ◽  
Fundamental Principle ◽  
Methanosarcina Acetivorans ◽  
Transport Pathway ◽  
Heterodisulfide Reductase ◽  
Genes Encoding ◽  
The Individual

ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2 -reducing methanogenic anaerobes (methanogens) from the domain Archaea . Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea . The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli , and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420 (F 420 H 2 ) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420 H 2 and reduction of ferredoxin with the exergonic oxidation of F 420 H 2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. IMPORTANCE Discovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2 -reducing methanogens to include diverse prokaryotes from the domains Bacteria and Archaea . The unprecedented coenzyme F 420 -dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4 -oxidizing pathways. The results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.

Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans

Biochemistry ◽  
2015 ◽  
Vol 54 (19) ◽  
pp. 3122-3128 ◽  
Author(s):  
Adepu K. Kumar ◽  
R. Siva Sai Kumar ◽  
Neela H. Yennawar ◽  
Hemant P. Yennawar ◽  
James G. Ferry
Keyword(s):  
Biochemical Characterization ◽  
Methanosarcina Acetivorans ◽  
Ferredoxin:Thioredoxin Reductase

Plant Pathology Diagnosed by X-Ray Microanalysis

1987 ◽  
Vol 45 ◽  
pp. 974-975
Author(s):  
J. H. Resau ◽  
N. Howell ◽  
S. H. Chang
Keyword(s):  
Electron Microscopy ◽  
Plant Pathology ◽  
Biochemical Characterization ◽  
Nutrient Deficiency ◽  
Green Leaf ◽  
Parasitic Infestation ◽  
X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

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