Recombinant Mannheimia succiniciproducens 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtnN)

What is the biochemical function of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase in bacterial metabolism?

5'-methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively . This enzyme plays a critical role in the metabolism of AdoHcy and MTA nucleosides in prokaryotic and lower eukaryotic organisms . The enzyme likely participates in the methionine salvage pathway in M. succiniciproducens, which is essential for recycling sulfur and maintaining proper cellular metabolism.

Methodological approach to study the function:

• Create gene knockouts using techniques similar to those employed in M. succiniciproducens studies

• Perform metabolite analysis to track accumulation of substrates and depletion of products

• Conduct comparative genomics with well-characterized mtnN enzymes from other organisms

• Use isotope labeling experiments to trace the metabolic fate of substrates

What are the optimal conditions for mtnN enzymatic activity and what methods are used to determine them?

While specific data for M. succiniciproducens mtnN is not directly available in the literature, related nucleosidases such as E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase function over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45°C typically being optimal .

To determine optimal conditions experimentally:

• Express and purify the recombinant enzyme using affinity chromatography

• Conduct activity assays across pH range 4.0-9.0 using appropriate buffer systems

• Test temperature optima between 25-60°C in 5°C increments

• Evaluate effects of various metal ions (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺) at concentrations of 1-10 mM

• Monitor activity using spectrophotometric assays that track the formation of adenine

How does the structure of M. succiniciproducens mtnN compare with the well-characterized E. coli enzyme?

The crystal structure of E. coli MTA/AdoHcy nucleosidase has been determined at 1.90 Å resolution using multiwavelength anomalous diffraction (MAD) . Each monomer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix . Intersubunit contacts between monomers are mediated primarily by helix-helix and helix-loop hydrophobic interactions .

While the specific structure of M. succiniciproducens mtnN has not been directly reported, structural characterization methodologies would include:

• Sequence alignment to identify conserved residues between the two enzymes

• Homology modeling using E. coli structure as template

• Validation through molecular dynamics simulations

• X-ray crystallography to resolve the actual structure

• Analysis of active site architecture and substrate binding pocket

What expression systems and purification strategies are most effective for producing active recombinant M. succiniciproducens mtnN?

Based on research with similar enzymes, the following approach is recommended:

Expression systems:

• E. coli BL21(DE3) with pET vectors for high yield

• Cold-adapted strains for improved protein folding

• Expression at OD₆₀₀ of 0.6-0.8 with IPTG (0.1-1.0 mM)

• Post-induction at 18-30°C for 4-24 hours

Purification strategy:

• Immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Ion exchange chromatography based on theoretical pI

• Size exclusion chromatography to obtain homogeneous preparations

• Addition of stabilizers (glycerol 10-20%) to maintain activity during storage

Quality assessment:

• SDS-PAGE for purity determination (>95%)

• Activity assays using synthetic substrates

• Mass spectrometry for identity confirmation

What are the standard kinetic parameters of M. succiniciproducens mtnN and how are they measured?

Standard kinetic parameters for nucleosidases typically include:

Methodological approaches:

• Use spectrophotometric assays that follow product formation

• Apply Michaelis-Menten kinetics to determine Km and Vmax

• Calculate kcat from Vmax and enzyme concentration

• Determine inhibition constants using appropriate inhibitors

• For comparison, the E. coli enzyme's potent inhibitor, 5'-(p-nitrophenyl)thioadenosine, has a Ki of 20nM

How does mtnN integration into M. succiniciproducens metabolic pathways influence succinic acid production?

M. succiniciproducens is known for efficient succinic acid production from various carbon sources . The integration of mtnN into its metabolic network likely affects succinic acid production through multiple mechanisms:

• Connections to central carbon metabolism:

  • Elementary mode analysis (EMC) has been used to gain insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production

  • mtnN activity could influence carbon flux similar to other metabolic enzymes

• Experimental approaches to investigate integration:

  • Construct mtnN gene knockout strains using techniques similar to those employed for other M. succiniciproducens genes

  • Perform metabolic flux analysis to determine changes in carbon flow

  • Conduct fed-batch fermentation to measure succinic acid production efficiency

• Potential interactions with other pathways:

  • zwf gene has been identified as a novel overexpression target for improved succinic acid production

  • NADPH-dependent mdh was selected for overexpression to synergistically improve succinic acid production

  • mtnN activity may influence these pathways through metabolite pool modifications

What molecular engineering strategies can enhance mtnN catalytic efficiency and substrate specificity?

Several engineering strategies can be employed to enhance mtnN properties:

• Structure-guided rational design:

  • Target active site residues based on structural analysis

  • Modify substrate binding pocket to improve affinity (lower Km)

  • Engineer catalytic residues to enhance turnover rate (higher kcat)

• Lessons from related enzymes:

  • A mutant of E. coli nucleosidase lacking the first 8 amino acids showed a Km approximately 3-fold higher than the full-length nucleosidase

  • This suggests N-terminal modifications might be explored for fine-tuning substrate affinity

• Domain-specific modifications:

  • The dual specificity for MTA and AdoHcy in the E. coli enzyme results from the truncation of a helix

  • Similar structural elements could be targeted for specificity engineering

• Screening and selection methodologies:

  • Develop high-throughput assays for detecting improved variants

  • Use competitive inhibition assays to evaluate binding improvements

  • Apply directed evolution with appropriate selection pressure

How do the kinetic parameters of wild-type versus engineered variants of M. succiniciproducens mtnN compare?

A comprehensive kinetic comparison would include:

Methodological approach:

• Conduct parallel characterization of wild-type and engineered variants

• Use identical experimental conditions for valid comparisons

• Apply multiple techniques (spectrophotometry, calorimetry, NMR) for comprehensive analysis

• Evaluate performance under physiologically relevant conditions

How can systems biology approaches integrate mtnN function with genome-scale metabolic models of M. succiniciproducens?

Systems biology integration requires several layers of analysis:

• Genomic integration:

  • Incorporate mtnN into existing genome-scale metabolic models of M. succiniciproducens

  • Map genetic regulatory networks affecting mtnN expression

• Transcriptional analysis:

  • Perform RNA-seq to measure mtnN transcript levels across growth phases

  • Compare with expression patterns of genes involved in succinic acid production

  • Analyze co-expression networks to identify functional relationships

• Metabolic flux analysis:

  • Apply elementary mode analysis (EMC) as previously used for M. succiniciproducens

  • Conduct 13C metabolic flux analysis to quantify intracellular reaction rates

  • Identify flux control coefficients to determine rate-limiting steps

• Integration with carbon source utilization:

  • M. succiniciproducens can efficiently utilize various carbon sources including sucrose

  • Investigate how mtnN activity varies with different carbon sources

  • Analyze interactions with phosphotransferase systems (PTS) used for sugar transport

What is the relationship between mtnN activity and the carbon metabolism during industrial fermentation processes?

The relationship between mtnN activity and carbon metabolism during fermentation involves several aspects:

• Carbon source-dependent expression:

  • M. succiniciproducens utilizes various carbon sources including sucrose, which requires specific phosphotransferase systems (PTS)

  • mtnN expression and activity may vary depending on the available carbon source

• Growth phase correlations:

  • Activity profiles should be mapped across batch and fed-batch processes

  • Changes in enzyme activity may correlate with shifts in metabolic flux

• Process optimization parameters:

  • Medium composition effects on mtnN activity and stability

  • Influence of pH, temperature, and dissolved oxygen on enzyme performance

  • Correlation between enzyme activity and succinic acid production yield

• Experimental approach:

  • Monitor enzyme activity throughout fermentation process

  • Track metabolite concentrations using chromatographic methods

  • Correlate enzyme activity with product formation rates

  • Apply metabolic control analysis to quantify control coefficients

What analytical methods are most sensitive for detecting mtnN activity in cell extracts?

Several analytical approaches provide varying degrees of sensitivity:

• Spectrophotometric assays:

  • Direct measurement of adenine formation at 260 nm

  • Coupled enzyme assays linking product formation to NAD(P)H oxidation/reduction

  • Colorimetric detection using specific reagents for reaction products

• Chromatographic methods:

  • HPLC separation and quantification of substrates and products

  • LC-MS/MS for highest sensitivity and specificity

  • Ion-exchange chromatography for separation of nucleosides and bases

• Radiometric assays:

  • Use of 14C or 3H-labeled substrates

  • Scintillation counting of separated products

  • Highest sensitivity for low activity samples

• Emerging technologies:

  • Biosensor-based detection systems

  • Fluorescence resonance energy transfer (FRET) assays

  • Isothermal titration calorimetry for thermodynamic parameters

How can crystallization conditions be optimized for structural determination of M. succiniciproducens mtnN?

Based on successful crystallization of related enzymes:

• Initial screening approaches:

  • Commercial sparse matrix screens (Hampton, Molecular Dimensions)

  • Grid screens around conditions successful for E. coli enzyme

  • Microseeding techniques to improve crystal quality

• Optimization variables:

  • Protein concentration (typically 5-20 mg/ml)

  • Precipitant type and concentration

  • pH range (focus on enzyme's stability optimum)

  • Temperature (4°C vs. 18°C vs. room temperature)

  • Additive screening for improved crystal formation

• Co-crystallization strategies:

  • Include substrates, products, or inhibitors

  • Try transition state analogs to capture catalytically relevant conformations

  • Use of heavy atom derivatives for phase determination

• Data collection considerations:

  • Cryoprotection optimization to minimize damage

  • Consideration of synchrotron radiation for highest resolution

  • Multiple wavelength anomalous dispersion (MAD) techniques as used for E. coli enzyme

What are the most effective gene knockout and complementation strategies for studying mtnN function in M. succiniciproducens?

Based on successful genetic manipulation of M. succiniciproducens:

• Gene knockout approaches:

  • Homologous recombination techniques similar to those used for MS0784, MS0807, MS0909, MS1233, and MS1237 gene knockouts

  • CRISPR-Cas9 based methods for precise genome editing

  • Selection markers appropriate for M. succiniciproducens

• Phenotypic analysis of knockouts:

  • Growth characteristics on different carbon sources

  • Metabolomic analysis to identify accumulated intermediates

  • Transcriptomic response to gene deletion

• Complementation strategies:

  • Plasmid-based expression under native or inducible promoters

  • Chromosomal integration at neutral sites

  • Heterologous expression of related mtnN genes from other organisms

• Experimental validation:

  • Enzyme activity assays to confirm loss and restoration of function

  • Metabolite profiling to verify pathway disruption and restoration

  • Growth phenotype analysis under various conditions

How might mtnN engineering contribute to improving industrial succinic acid production by M. succiniciproducens?

The potential for mtnN engineering to improve industrial applications involves several approaches:

• Metabolic pathway optimization:

  • Integration with existing metabolic engineering strategies

  • Synergistic effects with previously identified targets like zwf and mdh genes

  • Fine-tuning of metabolic flux through related pathways

• Fermentation process improvements:

  • Enhanced substrate utilization efficiency

  • Improved tolerance to inhibitory by-products

  • Maintained enzyme activity throughout extended fermentation periods

• Experimental design for industrial validation:

  • Scale-up studies from laboratory to pilot scale

  • Fed-batch fermentation optimization for engineered strains

  • Economic analysis of production improvements

• Integration with carbon source flexibility:

  • M. succiniciproducens can utilize various carbon sources including sucrose

  • Engineered mtnN might contribute to more efficient utilization of diverse feedstocks

  • Enhancement of metabolic pathways connected to different carbon uptake systems

What computational tools are most effective for predicting mutations that might enhance mtnN stability and activity?

Advanced computational approaches include:

• Structure-based computational methods:

  • Molecular dynamics simulations to identify flexible regions

  • Computational alanine scanning to determine energetic contributions

  • In silico docking for substrate binding optimization

  • Free energy calculations to predict stability changes

• Sequence-based approaches:

  • Multiple sequence alignment with related enzymes

  • Evolutionary conservation analysis

  • Correlated mutation analysis to identify co-evolving residues

  • Machine learning models trained on enzyme engineering datasets

• Combined approaches:

  • Rosetta protein design for activity and stability enhancement

  • FoldX for rapid stability predictions

  • PROSS (Protein Repair One Stop Shop) for stabilizing mutations

  • HotSpot Wizard for identification of catalytically important positions

• Validation methodologies:

  • In silico screening followed by targeted experimental validation

  • Iterative cycles of computation and experimental testing

  • Integration of structural and functional data into predictive models

How does mtnN activity correlate with stress response and adaptation in M. succiniciproducens?

Understanding the relationship between mtnN and stress adaptation requires:

• Stress exposure experiments:

  • Growth under various stressors (oxidative, acid, thermal)

  • Monitoring mtnN expression and activity during stress exposure

  • Recovery patterns after stress removal

• Mechanistic investigations:

  • Regulatory network analysis under stress conditions

  • Post-translational modifications affecting enzyme activity

  • Changes in substrate availability during stress response

• Experimental approaches:

  • Transcriptomics and proteomics under defined stress conditions

  • Enzyme activity assays to correlate gene expression with function

  • Metabolomic analysis to identify changes in related pathways

  • Growth and adaptation studies with wild-type and mtnN mutants

• Industrial process relevance:

  • Identification of stress factors affecting enzyme performance during fermentation

  • Development of strategies to maintain mtnN function under suboptimal conditions

  • Engineering stress-tolerant variants for improved industrial performance