Recombinant Mesoplasma florum NH (3)-dependent NAD (+) synthetase (nadE)

What is the genomic context of nadE in Mesoplasma florum?

The nadE gene in Mesoplasma florum is part of the organism's compact genome, which contains approximately 700 protein-coding genes. In the context of the iJL208 genome-scale metabolic model of M. florum, metabolic genes account for ~30% of the total gene count in the genome . The nadE gene would be among these metabolic genes, as NAD+ biosynthesis is critical for cellular energy metabolism. While specific location data is not provided in the search results, the gene likely resides in a region connected to other metabolic functions. Researchers studying this gene should analyze its context relative to flanking genes and its potential organization in operons, which could provide insights into regulatory mechanisms controlling its expression.

How does M. florum nadE differ from other bacterial NAD+ synthetases?

M. florum, as a near-minimal organism, has undergone significant genome reduction during its evolution. This implies that its nadE enzyme likely maintains only essential features required for functionality. Unlike some bacterial species that possess both NH(3)-dependent and glutamine-dependent NAD+ synthetases, M. florum appears to utilize specifically the NH(3)-dependent version. This specialization aligns with the metabolic streamlining observed in organisms with minimal genomes.

From a structural perspective, the NH(3)-dependent NAD+ synthetase from M. florum would be expected to contain the conserved ATP-binding domain and substrate recognition sites necessary for catalysis, but may lack regulatory domains or features present in larger, more complex bacterial species. Comparative genomic analysis would reveal specific sequence variations that could affect catalytic efficiency or substrate specificity optimized for M. florum's unique cellular environment.

What is the metabolic significance of nadE in M. florum's minimal metabolism?

In M. florum's streamlined metabolism, nadE plays a crucial role in maintaining NAD+/NADH balance, which is essential for numerous redox reactions in central metabolism. The iJL208 metabolic model of M. florum reveals that this organism relies heavily on glycolysis and fermentation pathways for energy production . The model shows M. florum can produce both lactate and acetate as fermentation products, with lactate production having a positive linear relationship with growth rate .

NAD+ regeneration is particularly critical in fermentative organisms like M. florum. Without a complete respiratory chain, the organism must regenerate NAD+ through fermentative pathways such as lactate production via lactate dehydrogenase (LDH: Mfl596), which was shown to have 4-8 fold higher expression compared to pyruvate dehydrogenase complex genes . The NAD+ produced by nadE would feed directly into these essential metabolic processes, making this enzyme an indispensable component of M. florum's core metabolism.

What expression systems are most effective for producing recombinant M. florum nadE?

When expressing recombinant M. florum nadE, researchers should consider several expression systems based on the specific experimental requirements:

E. coli Expression Systems:

• For high-yield production, E. coli BL21(DE3) or derivatives with pET vectors under T7 promoter control are recommended.

• Codon optimization may be necessary as M. florum uses a different codon preference than E. coli, particularly considering its low GC content as a member of the Mollicutes class.

• Expression at lower temperatures (16-20°C) may improve protein folding and solubility.

M. florum Expression Systems:

• For native conditions, expression within M. florum itself can be achieved using recently developed genetic tools.

• The oriC-based plasmids developed for M. florum that contain both the rpmH-dnaA and dnaA-dnaN intergenic regions would be suitable vectors .

• Selection can be performed using tetracycline resistance, as the tetM gene has been demonstrated to confer resistance to tetracycline at concentrations exceeding 100 μg/ml in M. florum .

• Transformation can be achieved through polyethylene glycol-mediated transformation (~4.1 × 10⁻⁶ transformants per viable cell) or electroporation (up to 7.87 × 10⁻⁶ transformants per viable cell) .

For either system, adding a purification tag (His6, GST, or MBP) is recommended, with careful consideration of whether N- or C-terminal placement would affect enzyme activity.

What purification protocol yields the highest activity for recombinant M. florum nadE?

A multi-step purification protocol optimized for M. florum nadE would include:

• Initial Capture: For His-tagged nadE, use immobilized metal affinity chromatography (IMAC) with a Ni-NTA column. Buffer conditions should include:

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 10% glycerol

  • Wash buffer: Same as lysis with 20 mM imidazole

  • Elution buffer: Same as lysis with 250 mM imidazole

• Intermediate Purification: Ion exchange chromatography (IEX) to separate charged variants:

  • Buffer A: 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 5% glycerol

  • Buffer B: Same as A with 1 M NaCl

  • Use a linear gradient from 5% to 100% Buffer B

• Polishing Step: Size exclusion chromatography:

  • Buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol

Throughout purification, maintain the temperature at 4°C and test fractions for activity using an NAD+ synthetase assay monitoring either ATP consumption or NAD+ formation. For optimal enzyme stability, include 1 mM DTT in all buffers and consider adding a metal cofactor (commonly Mg²⁺) if the enzyme shows metal-dependent activity.

How can I measure the kinetic parameters of M. florum nadE?

To determine the kinetic parameters of M. florum NH(3)-dependent NAD+ synthetase, use the following methodological approach:

Continuous Spectrophotometric Assay:

• Prepare reaction mixture containing:

  • 50 mM HEPES buffer (pH 7.5)

  • 10 mM MgCl₂

  • 1 mM ATP

  • Variable concentrations of NaAD (0.01-1 mM)

  • Variable concentrations of NH₄Cl (0.1-50 mM)

  • Purified recombinant nadE enzyme (10-100 nM)

• Monitor NAD⁺ formation by:

  • Direct measurement at 340 nm (ε = 6,220 M⁻¹cm⁻¹) or

  • Coupling the reaction with alcohol dehydrogenase and measuring NADH formation

• For determining Km and Vmax:

  • Vary NaAD concentration while keeping NH₄Cl constant at saturating levels

  • In separate experiments, vary NH₄Cl while keeping NaAD constant

  • Plot initial reaction rates versus substrate concentration

  • Fit data to Michaelis-Menten equation using non-linear regression

Expected Parameter Ranges:

For temperature and pH profiling, repeat the standard assay across different conditions (pH 5-10, temperature 15-45°C). When analyzing data, consider that as a near-minimal organism, M. florum may have evolved nadE with kinetic parameters that reflect its specialized metabolism and growth conditions.

How does M. florum nadE function within the context of the organism's genome-scale metabolic model?

The nadE enzyme functions as an integral component within M. florum's metabolic network as modeled in iJL208. This genome-scale metabolic model encompasses 370 reactions and accounts for approximately 30% of the protein-coding genes in M. florum's genome . The NAD+ produced by nadE feeds directly into key metabolic pathways:

• Glycolysis: NAD+ serves as an essential electron acceptor in the glyceraldehyde-3-phosphate dehydrogenase reaction, a critical step in glycolysis. The iJL208 model shows that glycolysis is a central pathway for M. florum energy generation .

• Fermentation Pathways: The model reveals that M. florum can produce both lactate and acetate as fermentation products, with an observed 8:1 lactate:acetate ratio . Lactate production through lactate dehydrogenase (LDH: Mfl596) directly consumes NADH and regenerates NAD+, creating a cyclic dependency on NAD+ availability.

• Redox Balance: Unlike organisms with complete respiratory chains, M. florum must maintain redox balance primarily through fermentation. The model indicates that acetate production requires oxygen to regenerate the NAD+ pool through NADH oxidase (NOX2, Mfl037) , highlighting the interconnection between oxygen availability, NAD+ regeneration, and metabolic flexibility.

To experimentally investigate nadE's role in this context, researchers could perform flux balance analysis using the iJL208 model with varying constraints on NAD+ synthesis rates, examining how perturbations in nadE activity would propagate throughout the metabolic network and affect predicted growth rates or metabolite secretion patterns.

What structural features of M. florum nadE contribute to its catalytic efficiency in a minimal organism?

While specific structural data for M. florum nadE is not provided in the search results, we can propose key structural features that likely contribute to its catalytic efficiency based on knowledge of NAD+ synthetases and M. florum's nature as a minimal organism:

• Domain Architecture: M. florum nadE likely preserves only essential structural domains required for catalysis, including:

  • An ATP-binding domain with conserved P-loop motif

  • A substrate binding pocket for NaAD

  • An ammonia binding site in the active center

  • Potentially simplified oligomerization interfaces

• Catalytic Residues: Key catalytic residues would be strictly conserved, including:

  • Lysine and arginine residues for ATP positioning

  • Aspartate residues for coordinating essential Mg²⁺ ions

  • Histidine and cysteine residues involved in substrate activation

• Structural Efficiency: As part of a minimal genome organism, M. florum nadE may exhibit:

  • Reduced size of non-catalytic regions

  • Optimized thermal stability for M. florum's growth conditions

  • Potentially enhanced substrate binding affinity to compensate for metabolic limitations

To experimentally investigate these features, researchers should consider:

• Performing site-directed mutagenesis of predicted catalytic residues

• Determining the oligomeric state using size exclusion chromatography and native PAGE

• Analyzing thermal stability through differential scanning fluorimetry

• Conducting crystallization trials to obtain a high-resolution structure

Comparing structural features with homologs from more complex organisms would provide insights into architectural elements specifically retained in this minimal organism context.

How could M. florum nadE be utilized for genome minimization studies?

M. florum nadE represents an excellent candidate for genome minimization studies due to M. florum's position as a near-minimal organism. Several research approaches could leverage this enzyme:

• Functional Reduction Analysis: Researchers could determine the minimal functional unit of nadE by:

  • Creating truncation variants to identify dispensable regions

  • Using deep mutational scanning to identify non-essential amino acids

  • Comparing with nadE from the minimal cell JCVI-syn3.0 (which contains only 531 genes)

• Integration with Genome Reduction Efforts: The iJL208 model has been used to propose an in silico reduced genome for M. florum containing 535 protein-coding genes . Researchers should:

  • Determine if nadE is included in this minimal gene set

  • Analyze how varying levels of nadE activity impact the viability of reduced genomes

  • Explore potential for further minimization of the nadE gene itself

• Synthetic Biology Applications: The genetic tools developed for M. florum, including oriC-based plasmids and transformation methods , could be used to:

  • Test synthetic variants of nadE with reduced functional complexity

  • Complement nadE knockouts with minimal synthetic versions

  • Investigate functional replacements with non-homologous NAD+ generating enzymes

This work would contribute to understanding the fundamental principles of minimal genomes and could help identify the essential functional components of NAD+ biosynthesis required for cellular life.

How should I address inconsistent activity in recombinant M. florum nadE preparations?

Inconsistent activity in recombinant M. florum nadE preparations can stem from multiple sources. Here's a methodical troubleshooting approach:

Common Issues and Solutions:

• Protein Stability Problems:

  • Monitor protein stability via SDS-PAGE and western blotting during storage

  • Test stabilizing additives: 10% glycerol, 1 mM DTT, 0.1 mM EDTA

  • Optimize storage conditions (typically -80°C with flash-freezing in small aliquots)

  • Consider storage at higher concentrations (>1 mg/ml) with carrier proteins like BSA (0.1 mg/ml)

• Cofactor Depletion or Contamination:

  • Ensure consistent Mg²⁺ concentrations (usually 5-10 mM) in all assays

  • Check for potential inhibitory metal contamination using ICP-MS

  • Dialyze protein preparations thoroughly before activity tests

  • Include metal chelation and reconstitution experiments to reset the active site

• Post-translational Modifications:

  • Analyze protein by mass spectrometry to detect modifications

  • Compare expression in different host systems (E. coli vs. native M. florum)

  • Test expression with different tags or tag positions if modifications occur near termini

• Experimental Design Control Table:

• Statistical Analysis:

  • Calculate the coefficient of variation (CV) between replicates (target: <15%)

  • Implement Grubbs' test to identify outliers in activity measurements

  • Use control charts to monitor enzyme activity trends over time

When working with M. florum nadE, which comes from a near-minimal organism, consider that the enzyme may have evolved to function optimally within M. florum's specific cellular environment and metabolic context , possibly requiring specific conditions not typically included in standard assay systems.

What approaches can resolve substrate specificity conflicts in published data on M. florum nadE?

When facing conflicting published data regarding substrate specificity of M. florum nadE, a systematic approach can help resolve discrepancies:

• Comprehensive Substrate Panel Testing:

  • Test all potential substrates under identical conditions:

    • NAD+ precursors: NaAD, nicotinic acid mononucleotide

    • Nitrogen sources: NH₃, NH₄⁺, glutamine

    • ATP analogs: ATP, dATP, GTP

  • Determine relative activity rates and Michaelis-Menten parameters for each

• Correlation with Structural Information:

  • Perform homology modeling based on related bacterial NAD+ synthetases

  • Identify substrate binding residues through docking simulations

  • Validate predictions through site-directed mutagenesis of key residues

  • Compare structural features with M. florum's metabolic capabilities as modeled in iJL208

• Consider Physiological Context:

  • Analyze metabolite concentrations in M. florum using metabolomics data

  • Examine nadE in the context of M. florum's minimal metabolism and fermentative lifestyle

  • Consider evolutionary pressures on substrate specificity in a near-minimal organism

• Technical Sources of Discrepancy:

  • Compare enzyme preparation methods across studies

  • Evaluate assay pH, temperature, and buffer conditions

  • Assess detection methods sensitivity and specificity

  • Consider the impact of different expression systems on enzyme properties

• Standardized Comparison Table:

This systematic approach not only addresses conflicts in published data but contributes to a more comprehensive understanding of M. florum nadE functioning within the context of a near-minimal organism.

How can I optimize transformation efficiency when expressing nadE constructs in M. florum?

Optimizing transformation efficiency for nadE constructs in M. florum requires attention to several key factors based on established M. florum genetic engineering techniques:

• Plasmid Design Considerations:

  • Include both rpmH-dnaA and dnaA-dnaN intergenic regions in oriC-based plasmids, as those containing only one region failed to produce detectable transformants

  • Maintain plasmid stability by ensuring appropriate spacing between these regions

  • Consider including a copy of the dnaA gene to potentially enhance replication efficiency

  • Optimize codon usage for M. florum's preference

• Transformation Method Selection:

  • Polyethylene glycol (PEG)-mediated transformation: Yields ~4.1 × 10⁻⁶ transformants per viable cell

  • Electroporation: Can reach frequencies up to 7.87 × 10⁻⁶ transformants per viable cell

  • Conjugation from E. coli: Achieves frequencies up to 8.44 × 10⁻⁷ transformants per viable cell

• Protocol Optimization for Electroporation:

  • Cell preparation: Harvest cells in exponential phase (OD600 0.4-0.6)

  • Washing buffer: 272 mM sucrose, 1 mM HEPES (pH 7.4)

  • DNA concentration: Use 1 μg of highly purified plasmid DNA per 100 μl cells

  • Electroporation settings: 25 μF capacitance as documented for M. florum

  • Recovery: Use rich media immediately after pulse application

• Selection Strategy:

  • Tetracycline resistance (tetM gene) has been demonstrated effective at concentrations exceeding 100 μg/ml

  • Other functional selectable markers include those conferring resistance to puromycin and spectinomycin/streptomycin

  • Use appropriate antibiotic concentrations based on MIC determination for wild-type M. florum

• Efficiency Optimization Table:

By systematically applying these strategies and carefully documenting outcomes, researchers can develop an optimized protocol for introducing nadE constructs into M. florum, facilitating studies of this essential enzyme in its native context.

How might M. florum nadE contribute to synthetic minimal genome projects?

M. florum nadE represents a valuable genetic element for synthetic minimal genome projects due to its origin in a near-minimal organism. Several promising research directions include:

• Comparison with Minimal Cell Models:

  • Analyze the nadE gene in M. florum versus the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0

  • Determine if the core functional domains are conserved across minimal genomes

  • Identify potential optimizations in the nadE sequence that contribute to genome minimization

• Essential Gene Set Integration:

  • The iJL208 model has been used to propose a minimal genome containing 535 protein-coding genes for M. florum

  • Determine if nadE is included in this minimal gene set and why

  • Investigate potential functional replacements or further simplifications of the nadE gene

• Chassis Development Applications:

  • Leverage the high growth rate of M. florum compared to other Mollicutes

  • Explore nadE's role in maintaining redox balance in minimal genome chassis organisms

  • Develop orthogonal NAD+ biosynthesis pathways for synthetic genome compartmentalization

• Engineering Optimization Strategies:

  • Create minimal functional versions of nadE through rational design

  • Explore potential for decreased genome size through enzyme promiscuity

  • Develop computational models to predict the impact of nadE variants on cellular fitness

The genetic tools already developed for M. florum, including oriC-based plasmids and various transformation methods , provide an excellent foundation for these investigations. The fact that M. florum has "no known pathogenic potential" also makes it an attractive organism for minimal genome engineering efforts compared to pathogenic Mycoplasma species that have been used in previous minimal genome studies.

What insights could comparative studies between M. florum nadE and homologs from other minimal organisms provide?

Comparative studies between M. florum nadE and homologs from other minimal organisms could yield valuable insights into essential enzyme features and evolutionary adaptations:

• Functional Conservation Analysis:

  • Compare M. florum nadE with homologs from:

    • JCVI-syn3.0 (531 genes, minimal synthetic Mycoplasma)

    • Mycoplasma genitalium (the smallest natural genome of any free-living organism)

    • Mycoplasma pneumoniae and Mycoplasma gallisepticum (other well-studied Mollicutes)

  • Identify conserved residues that represent the absolute minimal functional requirements

• Metabolic Context Comparison:

  • Analyze how nadE functions within different minimal metabolic networks:

    • Compare with the JCVI-syn3A metabolic model, which has different GAM (growth-associated maintenance) and NGAM (non-growth associated maintenance) values

    • Examine differences in substrate uptake rates and fermentation product profiles

    • Investigate correlation between nadE sequence variations and metabolic strategies

• Evolutionary Optimization Patterns:

  • Search for convergent evolution in nadE across independently reduced genomes

  • Identify adaptation signatures that correlate with specific metabolic constraints

  • Test whether observed variations represent optimization for specific cellular environments

• Comparative Parameter Table:

This comparative approach would contribute significantly to understanding the minimal functional requirements for NAD+ biosynthesis across different biological contexts and could inform future efforts in minimal genome design and synthetic biology.

How can structural studies of M. florum nadE inform the design of minimal synthetic enzymes?

Structural studies of M. florum nadE could provide critical insights for designing minimal synthetic enzymes with optimized functionality:

• Essential Structural Element Identification:

  • Determine high-resolution structure through X-ray crystallography or cryo-EM

  • Map the minimal catalytic core required for NH(3)-dependent NAD+ synthetase activity

  • Identify regions that could be further minimized without compromising function

  • Compare with the structural features of other enzymes in M. florum's metabolic network

• Structure-Guided Minimization:

  • Use computational approaches to predict the impact of specific deletions/mutations

  • Create a series of progressively reduced nadE variants maintaining critical catalytic residues

  • Test each variant for activity, stability, and kinetic parameters

  • Correlate structural features with the maintenance of function in M. florum's specific cellular environment

• Design Principles for Minimal Enzymes:

  • Analyze secondary structure elements essential for maintaining the active site geometry

  • Identify the minimal oligomerization interfaces required for function

  • Determine the contribution of specific domains to substrate specificity versus catalytic efficiency

  • Develop rules for maintaining protein stability with minimal sequence length

• Experimental Validation Approaches:

  • Express minimized variants in M. florum using established transformation methods

  • Test complementation of nadE deletion in vivo

  • Measure impact on growth rate and metabolic flux balance

  • Analyze effects on fermentation product profiles, which are known to be critical in M. florum metabolism

This research direction would not only advance our understanding of enzyme minimization principles but could also contribute to the broader field of synthetic biology by establishing design rules for creating efficient minimal enzymes for artificial cellular systems.

What are the key considerations for researchers beginning work with M. florum nadE?

Researchers initiating work with M. florum NH(3)-dependent NAD+ synthetase should consider several critical factors to ensure successful outcomes:

• Genetic and Metabolic Context:

  • M. florum represents a near-minimal organism with a streamlined metabolism suited for synthetic biology applications

  • nadE functions within a metabolic network where NAD+ regeneration is critical for maintaining redox balance in fermentation pathways

  • The genome-scale metabolic model iJL208 provides context for understanding how nadE integrates with other metabolic functions

• Technical Considerations:

  • Genetic manipulation can be achieved through PEG-mediated transformation, electroporation, or conjugation from E. coli

  • For plasmid-based expression, include both rpmH-dnaA and dnaA-dnaN intergenic regions for successful transformation

  • Select appropriate antibiotic resistance markers (tetracycline, puromycin, or spectinomycin/streptomycin) for selection

• Experimental Design Framework:

  • Begin with basic enzyme characterization (kinetics, substrate specificity, cofactor requirements)

  • Progress to structural studies to understand minimization potential

  • Advance to metabolic integration studies using the iJL208 model

  • Consider comparative studies with other minimal organisms to identify essential features

• Common Challenges and Solutions:

  • Low transformation efficiency can be addressed through optimized protocols

  • Protein solubility issues may require fusion tags or expression condition optimization

  • Physiological relevance requires consideration of M. florum's unique metabolic context, including its fermentative lifestyle and reliance on glycolysis

By approaching M. florum nadE research with these considerations in mind, investigators can effectively leverage this enzyme from a near-minimal organism to advance understanding of minimal gene sets and contribute to synthetic biology applications.

How does understanding M. florum nadE contribute to broader minimal genome research?

Understanding M. florum nadE provides valuable insights into minimal genome research through several interconnected avenues:

• Essential Gene Function Definition:

  • NAD+ biosynthesis represents a critical metabolic function preserved in minimal genome organisms

  • Studying nadE helps define the minimum functional requirements for this essential pathway

  • Comparison with the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 reveals key features of minimal gene sets

• Metabolic Network Integration:

  • The iJL208 genome-scale metabolic model of M. florum demonstrates how nadE fits within a minimal metabolic network

  • Understanding how NAD+ production connects to other pathways informs rational genome reduction approaches

  • The observed balance between lactate and acetate production pathways highlights essential metabolic flexibility even in minimal organisms

• Evolutionary Insights:

  • M. florum, with its reduced genome positioning it among the simplest free-living organisms , demonstrates natural genome minimization

  • nadE conservation reflects its indispensable role in cellular metabolism

  • Comparing nadE across Mollicutes with different genome sizes reveals evolutionary patterns in enzyme simplification

• Practical Applications:

  • The genetic tools developed for M. florum, including oriC-based plasmids and transformation methods , enable experimental validation of minimal genome designs

  • M. florum's high growth rate compared to other Mollicutes and lack of pathogenic potential make it an ideal chassis for synthetic biology applications

  • The in silico reduced genome prediction for M. florum (535 protein-coding genes) provides a roadmap for experimental genome minimization