Recombinant Nocardia farcinica Enolase-phosphatase E1 (mtnC)

What is Nocardia farcinica Enolase-phosphatase E1 (mtnC) and what is its biochemical function?

Nocardia farcinica Enolase-phosphatase E1 (mtnC) is a bifunctional enzyme involved in the methionine salvage pathway. The enzyme catalyzes two sequential reactions: first, the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) to form the intermediate 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate (HK-MTPenyl-1-P), and second, the dephosphorylation of this intermediate to produce the acireductone 1,2-dihydroxy-3-keto-5-methylthiopentene (DHK-MTPene). This dual functionality allows the enzyme to efficiently catalyze consecutive steps in a metabolic pathway that is crucial for recycling methionine and maintaining sulfur metabolism in the bacterium.

The enzyme is classified under EC 3.1.3.77 (2,3-diketo-5-methylthio-1-phosphopentane phosphatase) and is known by several synonyms including mtnC and NFA_45460. The full 218-amino acid sequence has been determined, with a specific target protein sequence that contributes to its catalytic properties.

How is Nocardia farcinica related to human disease, and why study its enzymes?

Nocardia farcinica is a clinically significant pathogenic bacterium within the Nocardia asteroides complex that causes nocardiosis, a serious infection affecting immunocompromised patients. This species is particularly important to differentiate from other Nocardia species because it characteristically demonstrates resistance to several extended-spectrum antimicrobial agents . Studies of N. farcinica virulence factors have shown that specific proteins facilitate invasion of host cells and interact with the host immune system.

Research on N. farcinica enzymes like mtnC provides insight into bacterial metabolism and potential virulence mechanisms. For example, other proteins from N. farcinica, such as Nfa34810, have been identified as immunodominant proteins located in the cell wall that facilitate bacterial invasion of host cells and trigger immune responses including the production of tumor necrosis factor alpha (TNF-α) . Understanding the metabolic enzymes of this pathogen could potentially reveal new therapeutic targets, especially given the antimicrobial resistance profile of N. farcinica.

What expression systems are used to produce recombinant N. farcinica mtnC?

Recombinant N. farcinica Enolase-phosphatase E1 can be expressed in multiple heterologous systems, each with distinct advantages for different research applications. According to the product information, this enzyme can be produced in various expression systems including:

• Yeast expression systems

• E. coli expression systems

• Baculovirus expression systems

• Mammalian cell expression systems

For research grade recombinant protein, the yeast expression system is commonly used as it provides appropriate eukaryotic post-translational modifications while maintaining high protein yields. For structural studies requiring large quantities of pure protein, E. coli systems may be preferred due to their high expression levels and simplified purification protocols. When native-like glycosylation patterns or other complex modifications are essential for functional studies, mammalian cell or baculovirus systems may provide advantages.

The choice of expression system should align with the specific research questions being addressed, considering factors such as protein solubility, yield, post-translational modifications, and downstream applications.

What role might mtnC play in N. farcinica pathogenesis and virulence?

While the direct role of mtnC in N. farcinica pathogenesis has not been explicitly established in the provided research results, parallels can be drawn from studies of other metabolic enzymes in bacterial pathogenesis. Metabolic enzymes often serve dual functions in bacterial pathogens - their primary metabolic role and secondary functions in virulence or immune evasion.

N. farcinica virulence involves the ability to invade host cells and modulate immune responses. The Nfa34810 protein of N. farcinica, for example, has been identified as an immunodominant protein located in the cell wall that facilitates bacterial invasion of host cells . It activates mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways in macrophages, leading to the production of TNF-α .

Enzymes in the methionine salvage pathway, including mtnC, may contribute to pathogenesis through:

• Nutrient acquisition during infection, particularly in nutrient-limited host environments

• Production of metabolites that may serve as signaling molecules

• Potential moonlighting functions in host-pathogen interactions

• Contribution to bacterial stress responses during host immune attack

Understanding whether mtnC plays a direct or indirect role in virulence would require genetic manipulation studies, such as creating deletion mutants (similar to the Δnfa34810 mutant described for studying Nfa34810 ) and assessing their ability to invade host cells or survive immune responses.

How does the structure and function of N. farcinica mtnC compare to homologous enzymes in other organisms?

Enolase-phosphatase enzymes are found across different domains of life, with varying degrees of sequence and structural conservation. In humans, enolase-phosphatase 1 (ENOPH1) has been studied particularly in the context of cancer, where its upregulation is associated with increased metastatic potential in hepatocellular carcinoma (HCC) .

A comparative analysis between bacterial mtnC and human ENOPH1 reveals several important aspects:

• Functional similarities: Both enzymes catalyze reactions in the methionine salvage pathway, though their specific roles may differ slightly between prokaryotes and eukaryotes.

• Pathological implications: While N. farcinica mtnC may contribute to bacterial metabolism during infection, human ENOPH1 has been implicated in cancer progression. ENOPH1 overexpression promotes cell migration and invasion in HCC, while ENOPH1 downregulation inhibits these processes .

• Signaling pathway interactions: ENOPH1 has been shown to enhance phosphorylation of AKT in human cancer cells , suggesting these enzymes may interface with signaling pathways. Similar interactions might exist for bacterial mtnC with bacterial signaling systems or with host signaling during infection.

These comparisons suggest that while core enzymatic functions may be conserved, the broader biological roles of these enzymes have evolved differently. For researchers, these similarities and differences offer opportunities to study fundamental aspects of enzyme evolution and specialization across different biological contexts.

What potential exists for mtnC as a therapeutic target against N. farcinica infections?

Targeting metabolic enzymes for antimicrobial development represents a promising approach, particularly for organisms like N. farcinica that exhibit resistance to conventional antibiotics. The potential of mtnC as a therapeutic target can be evaluated based on several factors:

• Essentiality: If mtnC is essential for N. farcinica survival or virulence, it would make an attractive target. Studies similar to those conducted for Nfa34810, where deletion mutants showed attenuated ability to infect cells , would be necessary to establish mtnC's importance.

• Structural uniqueness: The bifunctional nature of mtnC, catalyzing two consecutive reactions in the methionine salvage pathway, presents distinct targeting opportunities. Inhibitors could potentially disrupt either the enolization or phosphatase activities, or both.

• Selective targeting: An ideal therapeutic target allows for selective inhibition of the bacterial enzyme without affecting human homologs. Structural differences between bacterial mtnC and human ENOPH1 could potentially be exploited for selective inhibitor design.

• Pathway vulnerability: Disruption of the methionine salvage pathway might be particularly effective against N. farcinica in the nutrient-limited environment of host tissues during infection.

Development of potential inhibitors would require:

• High-resolution structural studies of mtnC

• Screening of compound libraries against recombinant mtnC

• Structure-activity relationship studies to optimize lead compounds

• Validation in cellular and animal infection models

The methionine salvage pathway has received limited attention as an antimicrobial target, suggesting this could be a novel approach for developing therapies against Nocardia infections.

What techniques are most effective for detection and identification of N. farcinica in clinical samples?

Accurate identification of N. farcinica is critical for proper diagnosis and treatment of nocardiosis. Traditional phenotypic methods are time-consuming and often lead to misidentification . Several molecular techniques have been developed that offer more rapid and reliable identification:

• PCR-based identification: A specific PCR assay using primers Nf1 and Nf2 has been developed for N. farcinica identification. This method amplifies a 314-bp fragment specific to N. farcinica . The PCR reaction includes:

  • PCR buffer: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂

  • Primers: 0.2 μM each of Nf1 and Nf2

  • Template: 1-5 μl genomic DNA

  • dNTPs: 0.2 mM each

  • Enzyme: 2.5 U of Taq polymerase

  • Total volume: 100 μl

• Restriction enzyme analysis: The 314-bp PCR products can be further analyzed by restriction enzyme digestion with CfoI, producing fragments of 218 bp and 78 bp (a third 18-bp fragment is typically not visible) . This provides additional confirmation of species identity.

• 16S rRNA gene analysis: Amplification and sequencing of the 16S rRNA gene can differentiate Nocardia species, though this may be less specific than targeted PCR approaches .

• RAPD analysis: Randomly amplified polymorphic DNA analysis has been used for both identification of Nocardia species and for epidemiological studies of N. farcinica .

For research involving mtnC, proper identification of the source organism is essential to ensure that the correct gene is being studied, particularly given the variability that can exist between different Nocardia species.

What assays can be used to measure the bifunctional activity of N. farcinica mtnC?

Measuring the dual enzymatic activities of mtnC requires assays that can detect both the enolization and phosphatase reactions. Here are methodological approaches for assessing each activity:

• Enolase activity assay:

  • Substrate preparation: Chemically synthesized 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P)

  • Reaction conditions: Typically performed at physiological pH (7.0-7.5) in appropriate buffer systems

  • Detection method: Spectrophotometric monitoring of the enolization reaction, which results in a shift in absorbance

  • Controls: Heat-inactivated enzyme as negative control

• Phosphatase activity assay:

  • Substrate: The intermediate product 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate (HK-MTPenyl-1-P)

  • Detection: Release of inorganic phosphate, which can be quantified using colorimetric methods such as the malachite green assay

  • Alternative approach: Using artificial phosphatase substrates like p-nitrophenyl phosphate, though these may not reflect native substrate specificity

• Coupled enzyme assay:

  • Complete reaction monitoring: Starting with DK-MTP-1-P and measuring the formation of the final product DHK-MTPene

  • Detection methods: HPLC or LC-MS for product formation

  • Kinetic analysis: Determination of kinetic parameters for both activities

• Isotope labeling approaches:

  • Using ³²P-labeled substrates to track phosphate release

  • ¹³C or ¹⁵N labeling to monitor carbon or nitrogen flow through the reaction pathway

  • Analysis by NMR spectroscopy or mass spectrometry

These assays would provide comprehensive characterization of mtnC activity and could be used to evaluate the effects of potential inhibitors or the impact of site-directed mutagenesis on enzyme function.

How can structural biology approaches inform our understanding of mtnC function?

Structural biology offers powerful tools for elucidating the molecular mechanisms of mtnC function. Several methodological approaches can provide complementary insights:

• X-ray crystallography:

  • Requires production of highly pure, homogeneous recombinant mtnC

  • Crystallization screening to identify conditions promoting crystal formation

  • Structure determination at high resolution (ideally <2.0 Å)

  • Co-crystallization with substrates, products, or inhibitors to capture different functional states

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful if crystallization proves challenging

  • Can reveal dynamic aspects of enzyme function

  • May provide insights into quaternary structure arrangements

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • For studying protein dynamics in solution

  • Examining substrate binding and conformational changes

  • Especially useful for mapping catalytic site interactions

• Computational approaches:

  • Homology modeling based on structures of related enolase-phosphatases

  • Molecular dynamics simulations to understand conformational flexibility

  • Docking studies to predict substrate binding modes and identify potential inhibitor binding sites

The structural data would enable:

• Identification of catalytic residues for each enzymatic activity

• Understanding how a single enzyme efficiently catalyzes two different reactions

• Design of site-directed mutagenesis experiments to probe structure-function relationships

• Rational design of specific inhibitors targeting either or both activities

Such structural studies would complement biochemical and genetic approaches to provide a comprehensive understanding of mtnC function in N. farcinica metabolism and potential pathogenesis.

How might studying mtnC contribute to understanding the metabolic adaptation of N. farcinica during infection?

Studying mtnC within the broader context of N. farcinica metabolism can provide insights into how this pathogen adapts to the host environment during infection. Several integrative approaches can be employed:

• Transcriptomic analysis: Examining mtnC expression levels under different conditions that mimic the host environment (nutrient limitation, oxidative stress, antimicrobial exposure) can reveal whether the methionine salvage pathway is upregulated during infection-relevant stresses. Similar approaches with other N. farcinica proteins have shown that virulence factors like Nfa34810 are expressed during infection and elicit antibody responses .

• Metabolomic profiling: Quantifying metabolites in the methionine salvage pathway in wild-type N. farcinica versus mtnC mutants would reveal the metabolic consequences of enzyme dysfunction. This approach has been valuable in hepatocellular carcinoma research, where metabolomic profiling revealed alterations in the cysteine/methionine metabolism pathway correlated with metastatic potential .

• In vivo infection models: Comparing the behavior of wild-type and mtnC-deficient N. farcinica strains in animal infection models could determine whether this enzyme contributes to in vivo survival and virulence, similar to studies conducted with other virulence factors .

• Systems biology integration: Combining datasets from genomics, transcriptomics, proteomics, and metabolomics can provide a comprehensive view of how mtnC functions within broader metabolic networks and adaptation strategies of N. farcinica.

This integrative approach could potentially identify metabolic vulnerabilities that could be exploited for therapeutic intervention, particularly given the challenges of antimicrobial resistance in N. farcinica infections.

What parallels exist between bacterial mtnC and the role of ENOPH1 in human disease?

The parallels between bacterial enolase-phosphatases like mtnC and human ENOPH1 present an intriguing opportunity for comparative research with potential translational implications:

• Functional conservation and divergence: Both enzymes participate in the methionine salvage pathway but may have evolved additional functions. Human ENOPH1 has been shown to promote cancer cell migration and invasion, whereas the potential moonlighting functions of bacterial mtnC remain to be explored .

• Signaling pathway interactions: ENOPH1 enhances AKT phosphorylation in hepatocellular carcinoma cells, suggesting a role in signaling beyond its catalytic function . Investigation of whether bacterial mtnC interacts with bacterial signaling systems or affects host cell signaling during infection could reveal novel aspects of host-pathogen interactions.

• Metabolic reprogramming: ENOPH1 upregulation is associated with metabolic alterations in cancer cells, particularly in the cysteine/methionine metabolism pathway . Similarly, mtnC may contribute to metabolic adaptation of N. farcinica during infection. Comparative metabolomic studies could identify conserved or divergent metabolic signatures.

• Therapeutic implications: Understanding the structure-function relationships across prokaryotic and eukaryotic enolase-phosphatases could inform drug discovery efforts for both infectious diseases and cancer. Compounds targeting conserved catalytic mechanisms might have dual applications, while differences might enable selective targeting.

This comparative approach highlights how fundamental research on bacterial enzymes can provide unexpected insights relevant to human disease and vice versa, emphasizing the value of cross-disciplinary research perspectives.

What are the key knowledge gaps and future research directions for N. farcinica mtnC?

Despite the available information on N. farcinica and its enzymes, several critical knowledge gaps remain regarding mtnC:

• Direct evidence of mtnC role in pathogenesis: Unlike Nfa34810, which has been demonstrated to facilitate N. farcinica invasion of host cells and trigger immune responses , the potential role of mtnC in virulence has not been directly established. Future research should include creation of mtnC deletion mutants and assessment of their virulence in cellular and animal models.

• Structural characterization: High-resolution structural data for N. farcinica mtnC would provide crucial insights into its bifunctional catalytic mechanism and facilitate inhibitor design. X-ray crystallography or cryo-EM studies should be prioritized.

• Metabolic network integration: Understanding how mtnC functions within the broader metabolic network of N. farcinica during infection would provide context for its potential importance. Metabolomic studies comparing wild-type and mtnC-deficient strains under infection-relevant conditions are needed.

• Host-pathogen interaction studies: Investigation of whether mtnC or its metabolic products interact with host cell processes, similar to how Nfa34810 activates MAPK and NF-κB signaling pathways , would reveal potential immunomodulatory roles.

• Drug discovery initiatives: Development and testing of selective inhibitors against N. farcinica mtnC could validate its potential as a therapeutic target and provide new treatment options for nocardiosis, especially important given the antimicrobial resistance characteristics of this species .