Recombinant Rhizobium leguminosarum bv. trifolii L-threonine 3-dehydrogenase (tdh)

Q: What is L-threonine 3-dehydrogenase and what reaction does it catalyze?

L-threonine 3-dehydrogenase (EC 1.1.1.103) is an oxidoreductase that catalyzes the NAD+-dependent oxidation of L-threonine to 2-amino-3-ketobutyrate (AKB). The reaction can be represented as:

L-threonine + NAD+ → 2-amino-3-ketobutyrate + NADH + H+

This enzyme belongs to the family of oxidoreductases acting on the CH-OH group of donors with NAD+ or NADP+ as an acceptor. The enzyme plays a key role in the threonine degradation pathway and participates in glycine, serine, and threonine metabolism .

Q: What is the biological significance of TDH in Rhizobium leguminosarum bv. trifolii?

In Rhizobium leguminosarum bv. trifolii, TDH contributes to the bacterium's metabolic versatility, which is critical for its symbiotic relationship with clover plants (Trifolium spp.). The enzyme participates in amino acid metabolism, which is essential for bacterial growth and adaptation to different environmental conditions. The threonine degradation pathway can supply acetyl-CoA and glycine, which are important metabolic intermediates for various cellular processes including energy production and biosynthesis .

Q: How does TDH from Rhizobium leguminosarum compare structurally to TDH from other organisms?

While detailed structural information specific to Rhizobium leguminosarum TDH is limited in the provided search results, comparative analysis can be made based on TDH from other organisms. TDH belongs to a family that includes short-chain (SDR), medium-chain (MDR), and long-chain (LDR) dehydrogenases/reductases. For instance, TDH from Trypanosoma brucei is a dimeric short-chain dehydrogenase that displays considerable conformational variation in its ligand-binding regions . In contrast, some bacterial TDHs like those from Clostridium difficile have been crystallized and structurally characterized, revealing specific active site configurations that are important for substrate binding and catalysis .

Q: What expression systems are recommended for recombinant production of R. leguminosarum TDH?

Based on research with similar enzymes, Escherichia coli is the preferred expression system for R. leguminosarum TDH. Specifically, E. coli BL21(DE3) has been successfully used for expressing similar dehydrogenases. The gene encoding TDH should be cloned into an appropriate expression vector containing a strong promoter (such as T7) and, ideally, a tag for purification (e.g., His-tag). For example, in a study with L-threonine aldolase from Pseudomonas putida KT2440, researchers successfully expressed the enzyme in E. coli BL21(DE3) .

Q: What is the most effective purification strategy for recombinant R. leguminosarum TDH?

A typical purification strategy would include:

• Affinity chromatography (if the recombinant protein includes a tag)

• Ion-exchange chromatography

• Size-exclusion chromatography

For optimal results, include protease inhibitors during cell lysis to prevent degradation. The purification buffer should contain components that stabilize the enzyme, potentially including:

• 50 mM Tris-HCl (pH 7.5-8.0)

• 150-300 mM NaCl

• 5-10% glycerol

• 1-5 mM DTT or β-mercaptoethanol

Based on studies with other dehydrogenases, maintaining temperature at 4°C throughout purification is crucial for preserving enzyme activity .

Q: How can I verify the identity and purity of recombinant R. leguminosarum TDH?

Multiple analytical methods should be used:

• SDS-PAGE to assess purity and determine molecular weight

• Western blot using antibodies against the tag or the enzyme itself

• Mass spectrometry for accurate molecular weight determination and peptide mapping

• Activity assay to confirm functional properties (monitoring NADH production spectrophotometrically at 340 nm)

• Circular dichroism to evaluate secondary structure integrity

Combining these methods provides comprehensive characterization of the recombinant enzyme's identity, purity, and structural integrity .

Q: What are the optimal conditions for measuring R. leguminosarum TDH activity?

While specific conditions for R. leguminosarum TDH are not explicitly provided in the search results, based on studies with similar enzymes, the following conditions are recommended:

• Buffer: 50-100 mM Tris-HCl or phosphate buffer

• pH: 7.5-8.5

• Temperature: 25-37°C

• NAD+ concentration: 1-2 mM

• L-threonine concentration: 10-50 mM

The reaction can be monitored by measuring the increase in absorbance at 340 nm due to NADH formation. Initial velocity measurements should be taken within the linear range of the enzyme activity .

Q: How can I determine the kinetic parameters (Km, Vmax, kcat) of R. leguminosarum TDH?

To determine kinetic parameters:

• Prepare a series of reaction mixtures with varying L-threonine concentrations (0.1-10× expected Km) while keeping NAD+ concentration constant and saturating.

• Similarly, prepare a series with varying NAD+ concentrations while keeping L-threonine constant and saturating.

• Measure the initial reaction rates for each substrate concentration.

• Plot the data using Lineweaver-Burk, Eadie-Hofstee, or non-linear regression methods to determine Km and Vmax.

• Calculate kcat from Vmax using the equation: kcat = Vmax/[E], where [E] is the total enzyme concentration.

For TDH from other organisms, Km values for L-threonine typically range from 0.1-10 mM, while Km values for NAD+ are generally in the micromolar range .

Q: What cofactors are required for R. leguminosarum TDH activity and how do they affect enzyme function?

R. leguminosarum TDH requires NAD+ as a primary cofactor for catalytic activity. Interestingly, based on research with related dehydrogenases from Rhizobium species, there might be flexibility in cofactor utilization. For instance, sulfolactaldehyde dehydrogenase (RlGabD) from R. leguminosarum SRDI565 can use both NAD+ and NADP+ as cofactors .

For optimal activity, the enzyme may also require divalent metal cations, as some TDHs from other organisms are metalloenzymes. The presence of these cofactors is crucial for proper active site geometry and efficient catalysis. The binding of NAD+ to the enzyme typically precedes substrate binding in an ordered mechanism, as observed in other dehydrogenases .

Q: What are the key active site residues in R. leguminosarum TDH and their roles in catalysis?

While the specific active site residues of R. leguminosarum TDH are not explicitly detailed in the search results, insights can be drawn from related TDHs. In TDH from Trypanosoma brucei, key residues involved in L-threonine binding include Ser82, Thr119, Thr186, Tyr144, and Trp280. The catalytic mechanism typically involves:

• A conserved tyrosine residue that acts as a catalytic base

• A serine or threonine residue that stabilizes the transition state

• Residues that position NAD+ correctly for hydride transfer

• Residues that bind and position the L-threonine substrate

These residues create a hydrogen-bonding network that orients the substrate for optimal catalysis, positioning the β-carbon of L-threonine for hydride transfer to the C4 atom of the nicotinamide ring of NAD+ .

Q: How can I improve the thermostability of recombinant R. leguminosarum TDH through protein engineering?

Based on successful approaches with related enzymes, the following strategies can be employed:

• Site-directed mutagenesis of key residues: Identify residues in the substrate-binding pocket for mutation. In a study with L-threonine aldolase from Pseudomonas putida, mutation of Asp93 to histidine (D93H) increased the melting temperature by 5°C (from 49.2°C to 54.2°C) .

• Molecular dynamics simulations: Use computational approaches to identify residues that might increase stability. Simulations can reveal how mutations affect the distance between key functional groups and substrate, potentially improving catalytic efficiency while enhancing thermostability .

• Introduction of disulfide bridges: Strategic placement of cysteine residues can form stabilizing disulfide bonds.

• Surface charge optimization: Modifying surface charges can improve solvent interactions and reduce aggregation at higher temperatures.

As demonstrated with L-threonine aldolase, mutations in the substrate-binding pocket can significantly improve both thermostability and catalytic efficiency .

Q: What protein domains are present in R. leguminosarum TDH and how do they contribute to enzyme function?

While specific domain information for R. leguminosarum TDH is not explicitly provided in the search results, TDH enzymes typically contain:

• Nucleotide-binding domain: Contains a Rossmann fold motif that binds NAD+ and is characterized by a βαβ structure.

• Substrate-binding domain: Contains residues that interact with L-threonine.

• Dimerization domain: Facilitates the formation of functional dimers, as observed in TDH from Trypanosoma brucei .

These domains work together to create the active site at their interface. The nucleotide-binding domain positions NAD+ correctly for the hydride transfer, while the substrate-binding domain orients L-threonine. Conformational changes upon substrate binding are often observed, which help align the reactants precisely for catalysis .

Q: How does TDH contribute to the symbiotic relationship between R. leguminosarum bv. trifolii and clover plants?

TDH plays an indirect but significant role in the symbiotic relationship by contributing to the metabolic versatility of R. leguminosarum bv. trifolii. This bacterium establishes symbiotic associations with clover plants (Trifolium spp.), where it fixes atmospheric nitrogen in exchange for carbon compounds from the plant .

The threonine degradation pathway, in which TDH is a key enzyme, generates metabolic intermediates that can feed into:

• Energy production pathways

• Biosynthesis of cellular components needed during nodule formation

• Production of metabolites that may influence plant-microbe signaling

Q: How is TDH expression regulated in R. leguminosarum bv. trifolii under different environmental conditions?

While specific information about TDH regulation in R. leguminosarum bv. trifolii is limited in the search results, insights can be drawn from studies on transcriptomic responses of this bacterium to various environmental conditions. Under stress conditions such as acidic pH and nutrient limitation, R. leguminosarum bv. trifolii undergoes significant transcriptional reprogramming, with differential expression of genes involved in amino acid metabolism .

TDH expression is likely regulated as part of the bacterium's response to:

• Carbon and nitrogen availability

• Oxygen concentration

• pH changes

• Symbiotic versus free-living states

Regulation may occur at transcriptional, post-transcriptional, and post-translational levels. Transcriptomic studies have shown that extrachromosomal replicons (ECRs) play a significant role in the stress response of R. leguminosarum bv. trifolii, suggesting that if the TDH gene is located on one of these replicons, its expression would be influenced by environmental conditions that trigger these stress responses .

Q: How does the threonine degradation pathway interact with other metabolic pathways in R. leguminosarum bv. trifolii?

The threonine degradation pathway, with TDH as a key enzyme, integrates with several other metabolic pathways in R. leguminosarum bv. trifolii:

• Glycine, serine, and threonine metabolism: TDH catalyzes the conversion of L-threonine to 2-amino-3-ketobutyrate, which is further converted to glycine and acetyl-CoA by 2-amino-3-ketobutyrate ligase (KBL) .

• TCA cycle: Acetyl-CoA generated from threonine catabolism can enter the TCA cycle for energy production.

• Fatty acid biosynthesis: Acetyl-CoA is a key precursor for fatty acid synthesis.

• One-carbon metabolism: Glycine produced from threonine degradation can enter one-carbon metabolism pathways.

• Aminoacetone metabolism: If 2-amino-3-ketobutyrate is not processed by KBL, it can spontaneously degrade to aminoacetone, which must be detoxified .

This metabolic interconnectivity allows R. leguminosarum bv. trifolii to efficiently utilize threonine as a carbon and nitrogen source, contributing to its adaptability in different environments, including during symbiotic association with host plants .

Q: How does R. leguminosarum TDH differ from TDH in other bacteria and eukaryotes?

Based on the search results, there are several notable differences between TDH from various organisms:

• Quaternary structure: TDH enzymes can exist as monomers, dimers, or tetramers. TDH from Trypanosoma brucei forms a dimer, while some bacterial TDHs may form tetramers .

• Cofactor preference: While most TDHs utilize NAD+ as the preferred cofactor, some show flexibility. For instance, a related dehydrogenase from R. leguminosarum can use both NAD+ and NADP+ .

• Substrate specificity: TDH from different organisms may show varying affinities for L-threonine and different sensitivity to inhibitors.

• Domain organization: TDHs can be classified as short-chain (SDR), medium-chain (MDR), or long-chain (LDR) dehydrogenases/reductases, with differences in domain organization and catalytic mechanism .

• Functionality in humans: Notably, TDH in humans is a non-functional pseudogene, which has implications for potential antimicrobial development targeting TDH in pathogens .

These differences highlight the evolutionary adaptations of TDH to fulfill specific metabolic requirements across different organisms.

Q: What can we learn from the crystal structures of TDH from other organisms that might apply to R. leguminosarum TDH?

Crystal structures of TDH from organisms like Trypanosoma brucei and Clostridium difficile provide valuable insights that may apply to R. leguminosarum TDH:

• Active site architecture: The structure of T. brucei TDH revealed that L-threonine interacts with specific residues (Ser82, Thr119, Thr186, Tyr144, and Trp280) through hydrogen bonds. The methyl group of L-threonine is oriented towards a hydrophobic residue (Trp280), while its β-carbon aligns with the C4 atom of the nicotinamide ring for hydride transfer .

• Conformational flexibility: T. brucei TDH displays considerable conformational variation in its ligand-binding regions, with a flexible loop (Loop 1) that closes over the active site when both NADH and L-threonine are bound .

• Binding mode of inhibitors: Structures with bound inhibitors (L-allo-threonine and pyruvate) show that these compounds occupy the L-threonine binding site, interacting with some of the same active site residues .

• Cofactor binding: The binding mode of NAD+ in the Rossmann fold provides insights into how this cofactor is positioned for catalysis.

These structural features likely have parallels in R. leguminosarum TDH and can inform experimental designs for enzyme characterization and engineering .

Q: What is the evolutionary relationship between TDH from R. leguminosarum and TDH from other organisms?

While the search results don't provide explicit phylogenetic analysis of TDH across different species, some inferences can be made:

TDH belongs to a family of dehydrogenases that includes short-chain (SDR), medium-chain (MDR), and long-chain (LDR) types, suggesting evolutionary divergence within this enzyme family. The presence of TDH in various prokaryotes and eukaryotes indicates an ancient evolutionary origin for this enzyme .

Specific evolutionary relationships might include:

• Conservation across bacteria: TDH is found in many bacterial species, including Rhizobium, Clostridium, and Pseudomonas, suggesting conservation of this metabolic function across diverse bacterial lineages .

• Loss of function in humans: Humans possess a TDH pseudogene that encodes a truncated, non-functional protein, indicating evolutionary loss of this pathway in the human lineage .

• Functional divergence: Related dehydrogenases in Rhizobium species show functional diversity, such as the ability to use different cofactors (NAD+ vs. NADP+), suggesting evolutionary adaptation to different metabolic requirements .

A comprehensive phylogenetic analysis would require sequence comparisons of TDH across multiple organisms to construct an evolutionary tree showing the relationships between TDH enzymes from different species.

Q: How can I design specific inhibitors for R. leguminosarum TDH for research purposes?

Designing specific inhibitors for R. leguminosarum TDH requires a structure-based approach:

• Use structural information: Though specific structural data for R. leguminosarum TDH isn't available in the search results, structures of related TDHs can guide inhibitor design. For example, the crystal structure of T. brucei TDH bound to inhibitors like L-allo-threonine and pyruvate shows how these compounds interact with active site residues .

• Focus on substrate analogs: Develop compounds that mimic L-threonine but contain modifications that prevent catalysis. Consider modifications to:

  • The hydroxyl group that is oxidized during the reaction

  • The amino group that forms hydrogen bonds with active site residues

  • The methyl group that interacts with hydrophobic pocket residues

• Target cofactor binding: Design compounds that interfere with NAD+ binding while maintaining specificity for TDH over other NAD+-dependent enzymes.

• Use molecular dynamics simulations: Employ computational approaches to predict how potential inhibitors interact with the enzyme and optimize their structures for improved binding.

• Test known inhibitors: Compounds like tetraethyl thiuram disulphide (TETD), which inhibits TDH from T. brucei, could serve as starting points for developing R. leguminosarum TDH inhibitors .

This approach can yield valuable research tools for studying the role of TDH in R. leguminosarum metabolism and symbiosis.

Q: What advanced techniques can be used to study the interaction between TDH and 2-amino-3-ketobutyrate ligase (KBL) in the threonine degradation pathway?

Several sophisticated techniques can be employed to study the TDH-KBL interaction:

• Protein-protein interaction assays:

  • Biolayer interferometry (BLI) or surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Fluorescence resonance energy transfer (FRET) with fluorescently labeled proteins

• Structural studies:

  • Cryo-electron microscopy to visualize the multi-enzyme complex

  • X-ray crystallography of co-crystallized TDH and KBL

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Functional studies:

  • Coupled enzyme assays to monitor substrate channeling between TDH and KBL

  • Site-directed mutagenesis of predicted interface residues to disrupt interaction

  • In vivo studies using bacterial two-hybrid systems or co-immunoprecipitation

• Computational approaches:

  • Molecular docking to predict interaction interfaces

  • Molecular dynamics simulations to study the dynamics of the complex

Evidence suggests that TDH and KBL may form a multi-enzyme complex to facilitate the channeling of the unstable intermediate 2-amino-3-ketobutyrate, preventing its spontaneous breakdown to aminoacetone and carbon dioxide . These techniques would help elucidate the structural basis and functional significance of this interaction.

Q: How can recombinant R. leguminosarum TDH be used in synthetic biology applications?

Recombinant R. leguminosarum TDH offers several promising applications in synthetic biology:

• Bioproduction of value-added compounds:

  • Pyrazine synthesis: TDH can supply aminoacetone, a precursor for pyrazine production. For example, 3-ethyl-2,5-dimethylpyrazine can be produced from L-threonine via a chemoenzymatic process involving TDH .

  • Production of specialized metabolites by integrating TDH into engineered metabolic pathways.

• Metabolic engineering:

  • Redirection of threonine metabolism to enhance production of glycine and acetyl-CoA

  • Creation of synthetic metabolic pathways that utilize TDH's catalytic abilities

  • Engineering of artificial enzyme cascades for novel compound synthesis

• Biosensors:

  • Development of NAD+/NADH ratio sensors based on TDH activity

  • Creation of threonine biosensors for metabolic engineering applications

• Protein scaffolding approaches:

  • Incorporating TDH into synthetic protein scaffolds with other enzymes to enhance metabolic flux

  • Creating synthetic multi-enzyme complexes that mimic the natural TDH-KBL interaction

• Directed evolution platforms:

  • Using R. leguminosarum TDH as a starting point for directed evolution experiments to create enzymes with novel activities or improved properties

These applications leverage the catalytic properties of TDH to create new synthetic biology tools and processes for research and biotechnology .

Q: What are common issues encountered when expressing recombinant R. leguminosarum TDH in E. coli and how can they be resolved?

When expressing recombinant R. leguminosarum TDH in E. coli, researchers may encounter several challenges:

• Poor expression levels:

  • Solution: Optimize codon usage for E. coli, try different promoters, or use specialized expression strains like BL21(DE3) Rosetta for rare codons.

  • Alternative: Lower induction temperature (16-25°C) and extend expression time.

• Protein insolubility/inclusion body formation:

  • Solution: Express at lower temperatures (16-20°C), use fusion tags that enhance solubility (SUMO, MBP, or TrxA), or add solubility enhancers to the culture medium.

  • Alternative: Develop refolding protocols from inclusion bodies if active enzyme can be recovered.

• Low enzyme activity:

  • Solution: Ensure proper cofactor addition during purification and activity assays.

  • Alternative: Try different buffer conditions and additives that might stabilize the enzyme.

• Protein instability during purification:

  • Solution: Include protease inhibitors, optimize buffer composition (add glycerol, reducing agents), and maintain low temperature throughout purification.

  • Alternative: Explore different purification strategies or shorten the purification time.

• Inconsistent kinetic parameters:

  • Solution: Ensure enzyme is fully saturated with cofactors, control temperature carefully during assays, and use fresh reagents.

  • Alternative: Compare assay results using different detection methods to identify potential interference.

These approaches have proven successful with similar enzymes and can be adapted specifically for R. leguminosarum TDH .

Q: How can I troubleshoot issues with TDH activity assays?

When troubleshooting TDH activity assays, consider the following approach:

• No detectable activity:

  • Verify enzyme integrity by SDS-PAGE and Western blot

  • Confirm NAD+ quality (NAD+ can degrade over time)

  • Test higher enzyme concentrations

  • Check pH and buffer composition

  • Ensure L-threonine is fresh and at appropriate concentration

• Low reproducibility:

  • Standardize assay temperature control

  • Prepare fresh reagents for each experiment

  • Use internal standards

  • Ensure consistent enzyme handling and storage

  • Monitor potential interfering substances in the assay

• Non-linear kinetics:

  • Check for product inhibition

  • Verify the assay is within the linear range of both enzyme concentration and reaction time

  • Look for potential substrate depletion

  • Test for potential allosteric effects

• High background absorbance:

  • Run appropriate blanks (without enzyme and/or substrate)

  • Check for spontaneous NAD+ reduction

  • Use high-purity reagents

  • Consider alternative detection methods if spectrophotometric interference persists

• Optimization strategy:

  • Systematically vary pH, temperature, buffer composition, and cofactor concentration

  • Compare continuous vs. endpoint assays

  • Consider coupled enzyme assays if the product is difficult to detect directly

Activity assays for TDH typically monitor NADH production at 340 nm, but alternative approaches such as fluorescence-based detection can offer greater sensitivity when needed .

Q: What strategies can help overcome the instability of the 2-amino-3-ketobutyrate intermediate in enzymatic studies?

The 2-amino-3-ketobutyrate (AKB) intermediate produced by TDH is unstable and can spontaneously degrade to aminoacetone and CO2, presenting challenges for enzymatic studies. To overcome this instability:

• Coupled enzyme assays:

  • Immediately couple TDH activity with 2-amino-3-ketobutyrate ligase (KBL) in the same reaction mixture

  • This mimics the natural system where these enzymes likely form a complex to channel the unstable intermediate

• Low-temperature studies:

  • Conduct experiments at reduced temperatures (4-10°C) to slow the spontaneous degradation

  • Optimize buffer conditions to enhance intermediate stability

• Rapid analysis techniques:

  • Use rapid-quench flow techniques to trap the intermediate

  • Employ mass spectrometry with direct infusion for real-time monitoring

• Chemical stabilization approaches:

  • Investigate chemical additives that might stabilize AKB

  • Consider derivatization strategies to trap the intermediate for analysis

• Computational predictions:

  • Use computational approaches to predict intermediate behavior

  • Model reaction kinetics accounting for the spontaneous degradation

• Alternative detection strategies:

  • Monitor aminoacetone formation as a proxy for AKB degradation

  • Develop specific detection methods for the products of the coupled TDH-KBL reaction

Research with similar unstable metabolic intermediates has shown that these approaches can significantly improve experimental outcomes and data reliability .