Toward the Use of Methyl-Coenzyme M Reductase for Methane Bioconversion Applications.

ConspectusAs the main component of natural gas and renewable biogas, methane is an abundant, affordable fuel. Thus, there is interest in converting these methane reserves into liquid fuels and commodity chemicals, which would contribute toward mitigating climate change, as well as provide potentially sustainable routes to chemical production. Unfortunately, specific activation of methane for conversion into other molecules is a difficult process due to the unreactive nature of methane C-H bonds.

Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F Modifications.

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F, a nickel-containing tetrahydrocorphin. Several modified versions of F have been discovered, including the 17-methylthio-F (mtF) used by ANME-1 MCR.

Control of biofilm formation by an pterin-binding periplasmic protein conserved among diverse .

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment.

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea.

Methylthiotransferases (MTTases) are radical -adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (msA) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein.

Control of Biofilm Formation by an Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment.

The Chlamydia trachomatis p-aminobenzoate synthase CADD is a manganese-dependent oxygenase that uses its own amino acid residues as substrates.

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen.

Highlighting the Unique Roles of Radical -Adenosylmethionine Enzymes in Methanogenic Archaea.

Radical -adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5'-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions.

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression.

Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by ) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F.

Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce β-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea.

Many methanogenic archaea synthesize β-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical -adenosylmethionine (SAM) aminomutases involved in the biosynthesis of N-acetyl-β-lysine and β-glutamate in C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from followed by biochemical characterization.

Biogenic formation of amorphous carbon by anaerobic methanotrophs and select methanogens.

Elemental carbon exists in different structural forms including graphite, diamond, fullerenes, and amorphous carbon. In nature, these materials are produced through abiotic chemical processes under high temperature and pressure but are considered generally inaccessible to biochemical synthesis or breakdown. Here, we identified and characterized elemental carbon isolated from consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which together carry out the anaerobic oxidation of methane (AOM).

An Unusual Route for -Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase.

lacks the canonical genes required for the biosynthesis of -aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from , the CT610 gene, that rescues Δ, Δ, and Δ mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site.

Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in .

Pterins are ubiquitous biomolecules with diverse functions including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen, A key player in this pathway is a pteridine reductase termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterize PruA to investigate the catalytic properties and substrate specificity of this pteridine reductase.

Identification of a Redox Active Thioquinoxalinol Sulfate Compound Produced by an Anaerobic Methane-Oxidizing Microbial Consortium.

The anaerobic oxidation of methane (AOM) mitigates the flux of methane from marine sediments into the water column. AOM is performed by anaerobic methanotrophic archaea (ANME) that reverse the methanogenesis pathway and partner bacteria that utilize the released reducing equivalents for sulfate reduction. Here, we investigated small-molecule extracts from sediment-free thermophilic enrichment cultures of ANME-1 and sulfate-reducing bacteria using ultraperformance liquid chromatography with high-resolution mass spectrometry.

Identification and biosynthesis of 2-(1H-imidazol-5-yl) ethan-1-ol (histaminol) in methanogenic archaea.

Histaminol is a relatively rare metabolite most commonly resulting from histidine metabolism. Here we describe histaminol production and secretion into the culture broth by the methanogen Methanococcus maripaludis S2 as well as a number of other methanogens. This work is the first identification of this compound as a natural product in methanogens.

Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F in Methanogens.

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F) moiety of coenzyme F in M.

Occurrence and biosynthesis of 3-mercaptopropionic acid in Methanocaldococcus jannaschii.

In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii, we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M.

A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens.

Unlabelled: The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin.

Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea.

The biosynthetic route for homocysteine, intermediate in methionine biosynthesis, is unknown in some methanogenic archaea because homologues of the canonical required genes cannot be identified. Here we demonstrate that Methanocaldococcus jannaschii can biosynthesize homocysteine from aspartate semialdehyde and hydrogen sulfide. Additionally, we confirm the genes involved in this new pathway in Methanosarcina acetivorans.

Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy.

The biochemical mechanism for the formation of the C-P-C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of the second C-P bond in l-phosphinothricin, we utilized a combination of stable isotopes and two-dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.

Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.

Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin.

Identification of structurally diverse methanofuran coenzymes in methanococcales that are both N-formylated and N-acetylated.

Methanofuran (MF) is a coenzyme necessary for the first step of methanogenesis from CO2. The well-characterized MF core structure is 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-γ-Glu2). Three different MF structures that differ on the basis of the composition of their side chains have been determined previously.

Discovery of multiple modified F(430) coenzymes in methanogens and anaerobic methanotrophic archaea suggests possible new roles for F(430) in nature.

Methane is a potent greenhouse gas that is generated and consumed in anaerobic environments through the energy metabolism of methanogens and anaerobic methanotrophic archaea (ANME), respectively. Coenzyme F430 is essential for methanogenesis, and a structural variant of F430, 17(2)-methylthio-F430 (F430-2), is found in ANME and is presumably essential for the anaerobic oxidation of methane. Here we use liquid chromatography-high-resolution mass spectrometry to identify several new structural variants of F430 in the cell extracts of selected methanogens and ANME.

Identification of a unique radical S-adenosylmethionine methylase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii.

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified.

Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis.

Fosfomycin is a broad-spectrum antibiotic that is useful against multi-drug resistant bacteria. Although its biosynthesis was first studied over 40 years ago, characterization of the penultimate methyl transfer reaction has eluded investigators. The enzyme believed to catalyze this reaction, Fom3, has been identified as a radical S-adenosyl-L-methionine (SAM) superfamily member.

In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea.

Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea.