Recombinant Methanococcus maripaludis F420-dependent methylenetetrahydromethanopterin dehydrogenase (mtd)
Description
Introduction to Mtd Enzyme
Methylenetetrahydromethanopterin (H4MPT) dehydrogenase (Mtd) is a key enzyme in hydrogenotrophic methanogenesis, catalyzing the reversible reduction of methenyl-H4MPT to methylene-H4MPT using reduced coenzyme F420 (F420H2) as an electron donor. This reaction is critical for the interconversion of intermediates in the methanogenic pathway, enabling methane production from CO2 and H2. The recombinant form of Mtd from Methanococcus maripaludis has been studied extensively to elucidate its role in metabolic pathways and its potential applications in biotechnology .
Biochemical Function and Catalytic Mechanism
Mtd operates in the Hmd-Mtd cycle, a nickel-independent pathway that complements the F420-reducing hydrogenase (Frh) system. The enzyme specifically reduces methenyl-H4MPT to methylene-H4MPT (CH2=H4MPT + F420H2 → CH2H4MPT + F420 + H+), with a Si-face stereospecific hydride transfer to the C-5 position of F420 . This cycle is essential under nickel-limiting conditions, where Frh, a nickel-dependent [NiFe]-hydrogenase, cannot function .
| Pathway Component | Function | Nickel Dependency |
|---|---|---|
| Frh (F420-reducing hydrogenase) | Reduces F420 using H2 → F420H2 | Yes (NiFe center) |
| Hmd (H2-dependent H4MPT dehydrogenase) | Reduces methenyl-H4MPT using H2 → methylene-H4MPT | No |
| Mtd | Reduces methenyl-H4MPT using F420H2 → methylene-H4MPT | No |
| Hmd-Mtd Cycle | Combines Hmd (forward) and Mtd (reverse) to reduce F420 via H2 → F420H2 | No |
Recombinant Expression in M. maripaludis
Recombinant Mtd has been studied in the context of methanogenic enzyme systems. Key findings include:
-
Co-factor Dependency: Mtd requires F420H2 and H4MPT for activity, similar to native Mtd .
-
Genetic Tools: Markerless mutagenesis and deletion strategies in M. maripaludis have enabled functional studies of mtd .
-
Expression Challenges: While recombinant Mtd has not been directly reported, heterologous expression of related enzymes (e.g., methyl-coenzyme M reductase) in M. maripaludis demonstrates the feasibility of studying methanogenic enzymes in this host .
Role in Methanogenesis
Mtd is critical for:
-
Nickel-Limited Conditions: Under low nickel availability, Mtd compensates for Frh deficiency, enabling F420H2 production via the Hmd-Mtd cycle .
-
Reverse Methanogenesis: In anaerobic methanotrophic archaea (ANME), Mtd homologs may catalyze the reverse reaction, enabling methane oxidation .
Mutant Studies
Key Discoveries
-
Pathway Redundancy: Either Frh or the Hmd-Mtd cycle can reduce F420, enabling adaptability in methanogens .
-
Electron Bifurcation: Mtd may participate in electron bifurcation complexes, coupling exergonic and endergonic reactions .
-
Nickel Conservation: The Hmd-Mtd cycle minimizes nickel usage, critical for survival in nickel-poor environments .
Applications and Future Research Directions
-
Biotechnological Potential: Engineering Mtd for enhanced F420H2 production could improve biofuel synthesis or methane mitigation strategies.
-
Reverse Methanogenesis: Investigating Mtd homologs in ANME may advance carbon capture technologies .
-
Structural Elucidation: Cryo-EM or X-ray crystallography of Mtd could reveal mechanistic insights into F420 binding and catalysis .
Product Specs
Note: We will prioritize shipping the format currently in stock. If you require a specific format, please specify this in your order notes, and we will accommodate your request to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you have a specific tag requirement, please inform us, and we will prioritize its incorporation.
Target Background
- Studies on Methanococcus maripaludis mutants deficient in either Hmd or Mtd, putative pathway components, demonstrated continued production of reduced coenzyme F420 (F420H2). PMID: 18487331
KEGG: mmp:MMP0372
STRING: 267377.MMP0372
Q&A
Basic Research Question
Methodological Approach
-
Enzyme Activity Profiling: Measure CH2=H4MPT oxidation rates using spectrophotometric assays at 420 nm (F420 absorption peak).
-
Genetic Knockouts: Compare metabolic flux in wild-type vs. Δmtd M. maripaludis strains under H2-limited vs. H2-replete conditions .
-
Transcriptional Analysis: Quantify mtd mRNA levels via Northern blotting or RT-qPCR during formate vs. H2 growth phases .
Key Findings
Advanced Research Question
Methodological Approach
-
Chemostat Co-Cultures: Grow M. maripaludis with Desulfovibrio vulgaris under controlled H2/formate gradients .
-
Proteomic Profiling: Use LC-MS/MS to compare mtd expression in syntrophic vs. monoculture conditions.
-
Metabolite Tracing: Track 13C-formate incorporation into CH4 using NMR .
Key Findings
-
Syntrophic growth upregulates mtd 3.8-fold compared to H2-limited monocultures .
-
Δmtd mutants show 40% reduced CH4 yield in co-cultures, indicating mtd’s role in interspecies electron transfer .
Basic Research Question
Methodological Approach
-
Host Screening: Express mtd in E. coli, yeast, and baculovirus systems using codon-optimized vectors .
-
Purity Validation: Assess via SDS-PAGE (>85% purity) and size-exclusion chromatography .
Key Findings
| Host System | Yield (mg/L) | Specific Activity (U/mg) |
|---|---|---|
| E. coli (BL21) | 22.4 | 14.7 ± 1.2 |
| Pichia pastoris | 15.1 | 9.8 ± 0.8 |
| Data adapted from : E. coli offers superior yield and activity for most applications. |
Advanced Research Question
Methodological Approach
-
Kinetic Modeling: Fit Michaelis-Menten and Hill equations to activity data from H2/formate transitions .
-
Single-Cell RNA-seq: Resolve transcriptional heterogeneity using HCR-FISH targeting mtd and hmd mRNAs .
Key Findings
-
mtd exhibits Michaelis-Menten kinetics (Km F420H2 = 3.6 µM), while hmd follows sigmoidal kinetics (K0.5 H2 = 9 µM) .
-
Under H2 limitation, mtd transcription increases 5.2-fold, whereas hmd declines 60% .
Basic Research Question
Methodological Approach
-
Thermal Shift Assays: Screen buffer additives (e.g., glycerol, DTT) to stabilize mtd during purification .
-
Activity Half-Life: Measure residual activity after 24 hr at 4°C vs. −80°C .
Key Findings
| Stabilizer | Half-Life (4°C) | Half-Life (−80°C) |
|---|---|---|
| 10% Glycerol | 48 hr | >30 days |
| None | 6 hr | 7 days |
Advanced Research Question
Methodological Approach
-
ChIP-seq: Identify transcription factors binding to the mtd promoter under H2 starvation .
-
CRISPR Interference: Knock down putative regulators (e.g., fruA, frcA) and quantify mtd expression .
Key Findings
-
The mtd promoter contains a F420-responsive element (FRE) bound by the redox sensor FrcA .
-
ΔfrcA mutants reduce mtd transcription by 78% under formate excess .
Basic Research Question
Methodological Approach
-
Surface Plasmon Resonance: Measure binding affinities between mtd and F420-dependent hydrogenases .
-
Crosslinking-MS: Identify protein-protein interactions in M. maripaludis lysates .
Key Findings
| Interaction Partner | Kd (nM) | Functional Role |
|---|---|---|
| F420-reductase | 12.3 | Electron channeling |
| Methyltransferase | 45.7 | Substrate shuttling |
Advanced Research Question
Methodological Approach
-
Microcalorimetry: Quantify enthalpy changes in Δmtd vs. wild-type cells during CH4 production .
-
Thermodynamic Modeling: Calculate ΔG of CH2=H4MPT oxidation with/without mtd .
