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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural and Mechanistic Advances in the Chemistry of Methyl-Coenzyme M Reductase (MCR)

Methane represents 34% of U.S. energy consumption and is a major greenhouse gas related to the global carbon cycle and energy production. However, current industrial practices significantly increase atmospheric methane levels, necessitating a deeper understanding of its biosynthesis and oxidation. Methyl-coenzyme M reductase (MCR) is central to biological methane metabolism, catalyzing the final step of methanogenesis and the first step in anaerobic methane oxidation. It is also a key target for strategies to capture and transform methane into value-added chemicals.The active site of MCR is a buried Ni-based cofactor only accessible by the substrates via a 50 Å long tunnel. Although the Ni(I) state is required to initiate catalysis, capturing this state remains a challenge for the current structural techniques. Recent advances in structural biology using X-ray Free-Electron Laser serial crystallography have provided insights into MCR's inactive Ni(II) state at room temperature and show promise for capturing its active Ni(I) form.Our team has established several critical aspects of the MCR mechanism using a combination of experimental and computational studies. MCR uses CH 3 -SCoM and CoBSH as substrates, producing methane and a disulfide product CoMSSCoB. Kinetic analysis showed that productive substrate binding requires CH 3 -SCoM to bind first, inducing conformational changes that optimize the active site for subsequent CoBSH binding. Following substrate binding, four proposed methane production/oxidation mechanisms were examined, establishing whether the reaction proceeds through an organometallic methyl-nickel(III), methyl anion ion, or methyl radical intermediate. Experimental measurements using CoBSH analogs successfully slowed the reaction, allowing for mechanistic insight that demonstrated the methyl radical pathway, where the initial interactions involve homolytic cleavage of the methyl-sulfur bond, generating a methyl radical that quickly abstracts the thiol hydrogen atom of CoBSH to form methane. Computational studies further confirmed that, compared to other mechanisms, the methyl radical mechanism is thermodynamically more favorable and accessible under physiological conditions.Spectroscopic and computational studies challenged the conventional understanding of substrate binding in MCR by proposing an alternative positioning of CH 3 -SCoM and CoMSSCoB in the active site pocket. The research suggested that CH 3 -SCoM (substrate) and CoMSSCoB (product) bind via their sulfonate groups to the Ni(I) center of cofactor F 430 . This binding allows for the reaction without substrate reorganization in the pocket but would require a long-range electron transfer. Altogether, the work summarized in this review reflects our current understanding of the enzyme's catalytic mechanism and structural dynamics. This is essential for developing efficient methane conversion technologies that could mitigate its environmental impact while harnessing energy-storage properties.

03 NATURAL GAS↗
DOE OSTI2025

A Mass Conservation Relaxed (MCR) LSTM Model for Streamflow Simulation Across CONUS

The recent development of the physics-aware Mass-Conserving Long Short-Term Memory network (MC-LSTM) provides an alternative to other data-driven Deep Learning (DL) models in hydrology. Mass-Conserving Long Short-Term Memory incorporates mass conservation directly into the LSTM architecture. Despite the theoretical advancements, studies have reported a surprisingly limited performance of the MC-LSTM in streamflow simulation. We hypothesize that such a limitation is due to the unrealistic mass conservation scheme in MC-LSTM, which overlooks unobserved incoming water fluxes beyond precipitation. As an attempt to verify this hypothesis, we propose a Mass Conservation Relaxed LSTM (MCR-LSTM), which incorporates a bi-directional mass relaxation (MR) component to account for potential incoming water fluxes beyond precipitation. We train and test the proposed MCR-LSTM model across 531 watersheds in the contiguous United States (CONUS) against three baseline models: the Sacramento Soil Moisture Accounting, LSTM, and MC-LSTM. Our results show that MCR-LSTM outperforms MC-LSTM despite its underperformance compared to LSTM. Specifically, MCR-LSTM's advantage over MC-LSTM is mainly seen in the Plains and Western U.S., where the newly incorporated MR component better simulates water loss and suggests the likely existence of additional incoming water fluxes beyond precipitation, respectively. The novelty and contribution of this study are twofold: firstly, it introduces an alternative physics-aware DL tool (i.e., MCR-LSTM) in hydrology with higher accuracy in specific regions compared to MC-LSTM. Secondly, it provides a diagnosis of regions where strict, precipitation-based mass conservation constraints may be unrealistic in streamflow simulation.

deep learning↗
NASA NTRS1983

Experiments on the retrieval of cloud parameters from the AMMS and MCR data

In the present experiment, Advanced Microwave Moisture Sounder (AMMS), Multispectral Cloud Radiometer (MCR) and lidar data collected during the Cooperative Convective Precipitation Experiment are combined in order to accomplish the retrieval of cloud top heights, temperatures, and integrated ice contents. It is demonstrated that the relationships among upwelling brightness temperatures obtained by AMMS channels are highly dependent on cloud properties. The retrieval of total ice content is achieved through the use of MCR and lidar data in determining cloud top height and temperature prior to the application of AMMS data.

Yeh, H.-Y. M.↗
NASA NTRS1973

Aerodynamic investigations on a 0.004 scale model MCR 0074 baseline space shuttle launch vehicle at Mach numbers between 0.6 and 4.96

A test of a 0.004-scale MCR 0074 Baseline Launch Configuration Space Shuttle model was conducted in the NASA-MSFC 14 x 14-inch Trisonic Wind Tunnel (MSFC TWT 566). The objective of the test was to determine the effects of model parametric variations on aerodynamic static stability characteristics over a Mach number range from 0.6 to 4.96. Angles-of-attack from minus 10 deg to plus 10 deg at 0 deg sideslip and angles-of-sideslip from minus 10 deg to plus 10 deg at minus 5 deg, 0 deg, and plus 5 deg angle-of-attack were investigated. The basic configuration investigated was the integrated vehicle consisting of the orbiter, and external tank, and two solid rocket boosters. It was designated 03T9S3.

Ramsey, P.↗
NASA NTRS1974

Triple balance test of the PRR baseline space shuttle configuration on a .004 scale model of the MCR 0074 orbiter configuration in the MSFC 14 x 14 inch Trisonic Wind Tunnel (TWT 570) IA31F(B), volume 1

A wind tunnel force and moment test of the space shuttle launch vehicle was conducted. The wind tunnel model utilized a triple balance such that component aerodynamics of the orbiter, external tank, and solid rocket booster was obtained. The test was conducted at an angle of attack range from -10 deg to 10 deg, and angle of sideslip range from -10 deg to 10 deg, and a Mach number range from 0.6 to 4.96. Simulation parameters to be used in future launch vehicle wind tunnel tests were investigated. The following were included: (1) effect of orbiter -ET attach hardware; (2) model attachment (spacer) effects; (3) effects of grit on model leading surfaces; and (4) model misalignment effects. The effects of external tank nose shape was studied by investigating five different nose configurations. Plotted and tabulated data is reported.

Ramsey, P. E.↗
NASA NTRS1975

An investigation in the NASA MSFC 14-inch trisonic wind tunnel to determine the pressure distribution over the components of a 0.004 scale version of the Rockwell MCR 0074 baseline shuttle ascent configuration (IA32F), volume 1

Data were obtained for Mach numbers from 0.6 to 3.48, angles of attack from -10 to 10 degrees, and angles of sideslip from -10 to 10 degrees at zero angle of attack. Also, -4 and 4 degrees sideslip were run for an angle of attack of -5 and 5 degrees. Aerodynamic configurations of the solid rocket motors, external tank, and orbiter are shown. Graphs of plotted pressure data (pressure coefficients) for the external tank and solid rocket motors are given. A description of the test facility is included.

Ramsey, P. E.↗
DOE OSTI2025

Colistin resistance plasmids dually enhance bacterial virulence and antibiotic resistance via surface polysaccharide biosynthesis

Plasmids carrying the mobilized colistin-resistance gene mcr-1 are prevalent among multidrug-resistant Gram-negative pathogens, yet their broad impact on bacterial physiology and virulence remains unclear. Here, we demonstrate that acquisition of an mcr-1 plasmid concurrently increases antimicrobial resistance and pathogenicity in Escherichia coli. On the same plasmid, the XRE-family transcriptional regulator EcaR cooperates with MCR-1 to activate the wec operon, driving biosynthesis of two surface polysaccharides: enterobacterial common antigen (ECA) and a high-molecular-weight O-chain. Expression of these surface polysaccharides increases bile resistance and virulence in a murine model and further elevates colistin resistance. MCR-1 enhances transcription of upstream genes in the wec operon, whereas EcaR directly activates an internal promoter (PwecE) to induce downstream gene expression. Thus, both components are required for surface polysaccharide expression, and deletion of either abolishes the phenotype. Genomic analysis of publicly available mcr plasmids reveals widespread co-occurrence of mcr-1 and ecaR on IncI2 and IncX4 plasmids, indicating their functional complementarity. These findings uncover a mechanism by which resistance plasmids remodel the bacterial surface, linking horizontal gene transfer to coordinated regulation of antimicrobial resistance and virulence.

Antimicrobial resistance↗
DOE OSTI2024

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis

Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel or other high-value commodity chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction that converts Ni-sirohydrochlorin a , c -diamide to 15,17 3 -seco-F430-17 3 -acid. This remarkable transformation, which involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c -acetamide side chain to form a γ-lactam ring, and the formation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes that catalyze multi-electron redox reactions. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, the following Specific Aims will be pursued to determine the: 1) Identity of the CfbCD reaction product. The exact reaction catalyzed by CfbCD, including the number of electrons transferred and whether it involves enzymatic cyclization, is unclear. Several approaches, including reaction stoichiometry measurements, spectroelectrochemistry, and magnetic resonance spectroscopy will be applied to elucidate the structure of the reaction product and establish whether CfbCD is a reductase or reductive cyclase. 2) Structure, conformational dynamics, and oligomerization state changes of CfbCD. Significant insight into the mechanism and allosteric regulation of CfbCD can be obtained by assessing changes in the structure and dynamics of the complex during the catalytic cycle. To accomplish this, a combination of size-exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and high-resolution structural methods will be employed. 3) Source, order, and stereochemistry of proton additions during CfbCD catalysis. Details regarding the order and stereochemistry of proton additions during the CfbCD reaction will be uncovered using a combined spectroscopic and computational approach. Complementary mechanistic studies employing site-directed mutagenesis and substrate analogs will establish the identity of active site acid residues and the possible involvement of substrate-assisted catalysis during the CfbCD reaction.

09 BIOMASS FUELS↗
DOE OSTI2025

Structure of active methyl-CoM reductase, Earth’s main methane producer

Summary Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the β-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

09 BIOMASS FUELS↗
DOE OSTI2025

Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase

ABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans , an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation. IMPORTANCE Methane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.

Rodriguez Carrero, Roy J. (ORCID:0000000184475641)↗
DOE OSTI2025

Use of Fisher's Ratio assisted multivariate curve resolution- alternating least squares for discovery-based analysis using ultrahigh pressure liquid chromatography-high resolution mass spectrometry

Non-targeted analysis of complex chemical mixtures can be difficult considering the convoluted nature of the matrix and the potential unknown chemical differences between samples or classes of samples. Ultrahigh pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) is an ideal technique to probe chemical differences for a wide variety of samples. While UHPLC-QTOF can discover minute chemical differences down to low part per billion (ppb) concentrations with a high degree of confidence, the application of high-resolution mass spectrometry can yield massive amounts of information (∼ 10 gb per sample) that cannot be analyzed manually. Therefore, the application of chemometric techniques is mandatory for the interrogation of complex samples. Fisher's ratio (FR) assisted multivariate curve resolution-alternating least squares (MCR-ALS) was used to the discover and identify the chemical differences between two classes of materials: 1) a pond water matrix and 2) the matrix spiked with a pharmaceutical standard mix containing 17 compounds. Thirteen of the seventeen spiked compounds were discovered using FR analysis, and then five were successfully deconvoluted using MCR-ALS wherein the number of curves chosen were automatically determined using singular value decomposition (SVD). In conclusion, the use of an automated FR assisted MCR-ALS will aid in discovering trace levels of chemical components without the need for the researcher to provide potentially biased input which will aid in non-targeted workflow.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Ecology of methyl-coenzyme M reductase encoding Thermoproteota

The recent demonstration that members of at least three classes of archaea affiliated with the Thermoproteota superphylum are involved in the production of the climate-active gas methane has sparked discussions about how well we understand the diversity of methanogens. Here, in this work, we show that members of all three of these lineages, as well as several other, yet uncultured and physiologically uncharacterized groups within the Thermoproteota that encode the key enzyme of anaerobic methane cycling, methyl-coenzyme M reductase (MCR), are widely distributed in anoxic ecosystems. We postulate that the taxonomic, metabolic, and ecological diversity of methanogenic and MCR-encoding Thermoproteota are poorly understood, and that the contribution of methylotrophic and thermoproteotal methanogenesis to methane production is largely unknown. We hypothesize that thermoproteotal methanogens could contribute, potentially substantially, to methane emissions in many anoxic environments that harbor methylated precursors, including wetlands, sediments, peat, rice paddies, wastewater sludge, and geothermal systems. We highlight the necessity to experimentally test the (eco)physiology of these widely distributed archaea using both culture-dependent ( in vitro ) and culture-independent ( in situ ) approaches to assess their potential contribution to methane emissions. Last, we stress the importance of remaining agnostic about the physiology of MCR-encoding Thermoproteota in the absence of experimental data because most of these archaea also carry the genetic potential to grow non-methanogenically.

MCR↗
DOE OSTI2024

The crystal structure of methanogen McrD , a methyl‐coenzyme M reductase‐associated protein

Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α 2 β 2 γ 2 ) enzyme responsible for methane formation via its unique F 430 cofactor. The genes responsible for producing MCR ( mcrA , mcrB and mcrG ) are typically colocated with two other highly conserved genes mcrC and mcrD . We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Preparation of coenzyme F430 biosynthetic enzymes and intermediates

Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. Here, the activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.

59 BASIC BIOLOGICAL SCIENCES↗