Recombinant Treponema denticola Fructose-bisphosphate aldolase class 1 (fda)

What is Treponema denticola Fructose-bisphosphate aldolase class 1 (fda)?

Treponema denticola Fructose-bisphosphate aldolase class 1 (fda) is an essential metabolic enzyme (EC 4.1.2.13) that catalyzes the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in the glycolytic pathway . It is a critical protein for T. denticola, a gram-negative, highly drug-resistant bacterium commonly found in primary dentition infections and periodontal disease . The enzyme consists of 295 amino acids and functions as a key component in both glycolysis and gluconeogenesis pathways . Unlike the Class II aldolases found in some other bacteria (such as M. tuberculosis), T. denticola fda belongs to Class I aldolases, which utilize a different catalytic mechanism that does not require metal cofactors .

How does T. denticola fda compare with aldolases from other organisms?

T. denticola fda belongs to Class I aldolases, which utilize a Schiff base mechanism involving a catalytic lysine residue. This contrasts with Class II aldolases (such as those from M. tuberculosis) that require zinc as a cofactor . Comparative studies of aldolases from various organisms reveal both similarities and differences in enzymatic properties:

Unlike the T. brucei enzyme, which shows characteristic deviation at low ionic strengths (below 0.1 M) where the apparent Km for Fru(1,6)P2 increases with decreasing salt concentration, detailed kinetic properties of T. denticola fda have not been fully characterized in the provided search results .

What role does T. denticola fda play in protein-protein interaction networks?

T. denticola fda participates in extensive protein-protein interaction (PPI) networks primarily associated with glycolysis pathways. According to STRING database analysis, fda interacts with 20 different proteins involved in carbohydrate metabolism . These interactions are represented as a network where fda appears as a central node (indicated in red in visualizations) connected to various neighboring proteins . The extensive connectivity suggests that fda plays a crucial role not only as an individual enzyme but also as part of a larger metabolic complex within T. denticola. Targeting fda could potentially disrupt multiple interconnected proteins, making it a valuable therapeutic target against this pathogen .

The interaction network includes proteins involved in:

• Glycolytic pathway enzymes

• Gluconeogenesis pathway components

• Energy metabolism regulators

• Sugar transporters and modifiers

This central position in metabolic networks highlights why fda has been identified as an essential protein for T. denticola survival and pathogenicity .

What are the potential applications of recombinant T. denticola fda in diagnostics?

While specific diagnostic applications for T. denticola fda have not been directly addressed in the search results, fructose-1,6-bisphosphate aldolase from other pathogens has shown significant potential as a diagnostic tool. For instance, FBPA from Schistosoma japonicum has been successfully used as an antigen for immunodiagnosis of infection in water buffaloes, with 100% specificity and 100% sensitivity in ELISA tests . Similarly, FBPA has demonstrated utility in the immunodiagnosis of malaria .

The potential diagnostic applications of T. denticola fda could include:

• Development of serological assays to detect antibodies against T. denticola in patients with periodontal disease

• Creation of molecular diagnostic tools targeting fda gene expression in clinical samples

• Use as a biomarker for antimicrobial resistance profiling

Given that T. denticola is a highly drug-resistant bacterium found in dental infections, recombinant fda could serve as a valuable tool for early detection and monitoring of these infections .

How might inhibitors of T. denticola fda be developed as antimicrobial agents?

Developing inhibitors against T. denticola fda represents a promising antimicrobial strategy based on several factors:

• Essential metabolic role: As fda is crucial for central carbon metabolism in T. denticola, inhibiting its function would likely have significant impacts on bacterial viability .

• PPI network centrality: The extensive protein-protein interaction network centered around fda suggests that its inhibition could disrupt multiple metabolic pathways simultaneously .

• Structural distinctiveness: Class I aldolases like T. denticola fda utilize a Schiff base mechanism, which differs from human Class I aldolases in specific active site architectures that could be exploited for selective targeting .

• Drug target validation: In silico analysis has identified fda as one of 11 essential proteins in T. denticola that could serve as valuable therapeutic targets .

Potential approaches for inhibitor development include:

• Structure-based design targeting the active site

• Allosteric inhibitors disrupting enzyme dynamics

• Covalent modifiers targeting the catalytic lysine residue

• Peptide-based inhibitors disrupting protein-protein interactions

The development process would benefit from the availability of high-quality recombinant protein (>85% purity by SDS-PAGE) for high-throughput screening and structural studies .

What are the optimal conditions for expressing and purifying recombinant T. denticola fda?

Based on the available information and experience with similar aldolases, the following methodological approach is recommended for optimal expression and purification of recombinant T. denticola fda:

Expression System and Conditions:

• E. coli is the preferred expression host, as successfully demonstrated for T. denticola fda .

• Caution should be exercised with N-terminal fusion tags, as these may result in inactive, mostly insoluble protein (as observed with M. tuberculosis aldolase) .

• C-terminal 6His-tagging appears to be compatible with enzyme activity and can facilitate purification .

• Expression should be conducted at lower temperatures (16-25°C) to enhance proper folding.

Purification Protocol:

• Lyse cells in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Clarify lysate by centrifugation at 20,000 × g for 30 minutes at 4°C.

• For His-tagged protein, purify using Ni-NTA affinity chromatography.

• Further purify by ion-exchange chromatography followed by size-exclusion chromatography.

• Assess purity by SDS-PAGE (target >85%) .

Storage Recommendations:

• For liquid formulations, store at -20°C/-80°C with an expected shelf life of 6 months .

• For lyophilized formulations, store at -20°C/-80°C with an expected shelf life of 12 months .

• Avoid repeated freeze-thaw cycles .

• Working aliquots can be stored at 4°C for up to one week .

• For long-term storage, add glycerol to a final concentration of 5-50% (recommended default: 50%) .

How can the enzymatic activity of T. denticola fda be measured?

The enzymatic activity of T. denticola fda can be assessed through several complementary approaches based on its role in catalyzing both the forward and reverse reactions of fructose 1,6-bisphosphate cleavage:

Forward Reaction (Fru(1,6)P2 cleavage) Assay:

• Coupled spectrophotometric assay:

  • Mix purified enzyme with fructose 1,6-bisphosphate in appropriate buffer.

  • Include coupling enzymes (triose phosphate isomerase and glycerol-3-phosphate dehydrogenase) and NADH.

  • Monitor NADH oxidation at 340 nm as a measure of dihydroxyacetone phosphate production.

  • Calculate specific activity in U/mg of protein .

• pH optimum determination:

  • Perform activity assays across a range of pH values (6.0-9.0).

  • T. denticola fda likely exhibits a broad pH optimum for Fru(1,6)P2 cleavage similar to other Class I aldolases .

Alternative Substrate (Fru-1-P) Assay:

• Similar to the forward reaction assay but using fructose 1-phosphate as substrate.

• Class I aldolases typically show a narrower pH optimum for Fru-1-P cleavage compared to Fru(1,6)P2 cleavage .

Reverse Reaction (Aldol Condensation) Assay:

• Mix enzyme with dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

• Monitor formation of Fru(1,6)P2 using coupled enzymatic assays or HPLC analysis.

The specific activity of recombinant fda from T. denticola has not been explicitly stated in the search results, but for comparison, the specific activity of M. tuberculosis Class II aldolase is reported as 35 U/mg, approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen .

What approaches can be used to study T. denticola fda structure-function relationships?

Understanding the structure-function relationships of T. denticola fda requires a multidisciplinary approach combining computational, biophysical, and biochemical methods:

Computational Methods:

• Homology modeling:

  • Generate 3D structure models using online servers like SWISS-MODEL .

  • Validate models using tools such as PROCHECK, VERIFY3D, and ERRAT.

  • Compare with crystal structures of homologous Class I aldolases.

• Molecular dynamics simulations:

  • Investigate protein flexibility and conformational changes during catalysis.

  • Study substrate binding and product release pathways.

  • Identify potential allosteric sites for inhibitor design.

Experimental Structure Determination:

• X-ray crystallography:

  • Crystallize purified protein (>85% purity) under various conditions .

  • Collect diffraction data and solve structure.

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

• NMR spectroscopy (for specific domains or peptides):

  • Study protein dynamics in solution.

  • Analyze substrate binding and conformational changes.

Functional Analysis:

• Site-directed mutagenesis:

  • Target catalytic residues (likely lysine residues involved in Schiff base formation).

  • Modify residues at the active site or protein-protein interaction interfaces.

• Kinetic analysis:

  • Determine Km, kcat, and kcat/Km for wild-type and mutant enzymes.

  • Assess substrate specificity and inhibitor sensitivity.

  • Study pH dependence and ionic strength effects on enzyme activity .

• Protein-protein interaction studies:

  • Validate predicted interactions using pull-down assays, co-immunoprecipitation, or surface plasmon resonance.

  • Map interaction surfaces using protein fragments or peptide arrays.

Through these complementary approaches, researchers can develop a comprehensive understanding of how T. denticola fda structure relates to its catalytic function, substrate specificity, and role in protein-protein interaction networks.