Recombinant Cupriavidus taiwanensis Triosephosphate isomerase (tpiA)

What is Triosephosphate isomerase (TpiA) and what is its role in Cupriavidus taiwanensis metabolism?

Triosephosphate isomerase (TpiA) is a key glycolytic enzyme that catalyzes the reversible interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. This isomerization reaction is critical for efficient energy production and carbon utilization in bacteria. In C. taiwanensis, as in other bacteria, TpiA plays several essential metabolic roles:

TpiA facilitates energy production through glycolysis, provides precursors for the biosynthesis of amino acids and lipids, and prevents the accumulation of dihydroxyacetone phosphate, which could otherwise lead to the formation of toxic methylglyoxal . The enzyme's activity is essential for maintaining metabolic homeostasis, which influences various cellular processes including growth, potential virulence factors, and response to environmental stresses .

Experimental evidence from studies in Pseudomonas aeruginosa has demonstrated that TpiA influences both bacterial metabolism and antibiotic resistance mechanisms, suggesting similar functional importance in other bacterial species including C. taiwanensis .

What structural characteristics define Cupriavidus taiwanensis TpiA and how do they relate to its function?

Triosephosphate isomerases, including C. taiwanensis TpiA, typically display a characteristic (β/α)8 barrel or TIM-barrel fold. Key structural features include:

• A dimeric quaternary structure that functions as an obligate dimer, essential for optimal catalytic activity

• An active site composed of three invariable catalytic residues (Lysine, Histidine, and Glutamate)

• A substrate binding pocket that accommodates the phosphate moiety

• Loop regions that undergo conformational changes during catalysis

Each monomer contributes residues to complete the active site of the adjacent monomer, meaning that disruption of the dimeric structure directly impairs catalytic function. The evolutionary conservation of these structural elements across bacterial species highlights their importance for enzymatic function .

What expression systems and purification strategies are recommended for producing recombinant C. taiwanensis TpiA?

For optimal expression and purification of recombinant C. taiwanensis TpiA, the following methodological approach is recommended:

Expression System Selection:

• Escherichia coli BL21(DE3) remains the preferred host for high-yield expression

• Consider codon optimization if C. taiwanensis codons differ significantly from E. coli

• Recommended vectors include pET series with N-terminal or C-terminal His-tags

Expression Optimization Parameters:

Purification Protocol:

• Cell lysis using sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

• Immobilized metal affinity chromatography using Ni-NTA resin

• Size exclusion chromatography to separate dimeric active enzyme from aggregates

• Activity verification using coupled spectrophotometric assay

The purification strategy should yield protein with >95% purity as assessed by SDS-PAGE with typical yields of 15-30 mg per liter of bacterial culture.

How can researchers assess the enzymatic activity and kinetic parameters of recombinant C. taiwanensis TpiA?

Several complementary approaches can be used to assess the enzymatic activity and kinetic parameters of recombinant C. taiwanensis TpiA:

Spectrophotometric Coupled Assay Protocol:

• Prepare reaction mixture containing 100 mM Tris-HCl (pH 7.4), 0.2 mM NADH, and 1 unit/mL of glycerol-3-phosphate dehydrogenase

• Add purified TpiA (0.1-1.0 μg) to the mixture

• Initiate reaction by adding D-glyceraldehyde-3-phosphate (0.05-5.0 mM)

• Monitor decrease in NADH absorbance at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

• Calculate activity as μmol of substrate converted per minute per mg of protein

Determination of Kinetic Parameters:

• Generate Michaelis-Menten curves by varying substrate concentration

• Calculate Km and Vmax using non-linear regression analysis

• Determine kcat by dividing Vmax by enzyme concentration

• Calculate catalytic efficiency as kcat/Km

Typical Kinetic Parameters for Bacterial TPIs:

These methodologies provide quantitative assessment of enzyme activity and allow comparison with TpiA enzymes from other bacterial species.

How might TpiA contribute to the metabolic adaptability and potential virulence of C. taiwanensis?

Based on studies of TpiA in other bacterial species, particularly P. aeruginosa, this enzyme likely plays important roles in C. taiwanensis adaptability and potential virulence mechanisms:

TpiA's central position in glycolysis makes it critical for energy generation during various growth phases and environmental conditions. Recent research has demonstrated that TpiA is a key metabolic enzyme affecting virulence and antibiotic resistance in P. aeruginosa, suggesting similar functional importance in other bacteria including C. taiwanensis .

The metabolic flexibility conferred by functional TpiA allows bacteria to adapt to varying nutrient conditions, which is particularly important during host colonization or environmental transitions. Additionally, the ATP generated through glycolysis provides energy for the synthesis and secretion of potential virulence factors .

Experimental approaches to investigate these roles include:

• Generating tpiA knockout or knockdown mutants in C. taiwanensis and assessing phenotypic changes

• Metabolomic profiling to identify shifts in central carbon metabolism

• Transcriptomic analysis to identify genes co-regulated with tpiA under different conditions

• Comparative studies with known symbiotic and pathogenic C. taiwanensis strains

What role might TpiA play in the symbiotic relationship between C. taiwanensis and leguminous plants like Mimosa pudica?

C. taiwanensis is known to form symbiotic relationships with leguminous plants such as Mimosa pudica . TpiA likely contributes to this symbiotic relationship through multiple metabolic pathways:

During nodule formation and nitrogen fixation, bacterial metabolism must adapt to the microaerobic environment within the nodule. TpiA's role in glycolysis would be crucial for generating energy under these conditions. Additionally, the carbon skeletons produced through glycolysis serve as precursors for amino acid biosynthesis, which is essential during symbiotic nitrogen fixation .

The expression of ACC deaminase in C. taiwanensis has been shown to increase nodulation and plant growth promotion in Mimosa pudica . The metabolic pathways involving TpiA likely interact with ACC deaminase activity, as both influence central carbon metabolism and energy production during symbiosis.

Research approaches to investigate this connection include:

• Creating tpiA mutants and assessing their nodulation efficiency

• Metabolic flux analysis during different stages of symbiotic interaction

• Transcriptomic analysis comparing tpiA expression in free-living versus symbiotic states

• Co-expression analysis of tpiA with known symbiosis genes

How do mutations in the active site of C. taiwanensis TpiA affect its catalytic efficiency and substrate specificity?

Mutations in the active site of C. taiwanensis TpiA can significantly impact its function through several mechanisms:

The active site of TpiA contains three invariable catalytic residues (Lysine, Histidine, and Glutamate) that are essential for the isomerization reaction . Mutations in these conserved residues typically result in dramatic reduction or complete loss of enzymatic activity. For example, studies on TPIs from parasitic organisms have shown that modifications in the active site can decrease catalysis, making these enzymes potential drug targets .

Effects of Specific Active Site Mutations:

The dimeric structure of TpiA is crucial for its function, and mutations at the dimer interface can destabilize the quaternary structure, typically reducing catalytic efficiency .

How does the genetic context of the tpiA gene in the C. taiwanensis genome influence its expression and regulation?

The genomic context of tpiA in C. taiwanensis could significantly influence its expression and regulation:

Analysis of the C. taiwanensis genome reveals important insights into the genetic organization around the tpiA gene. In C. taiwanensis strain STM 6018, the genome contains various secretion systems and protein secretion mechanisms that may interact with or influence tpiA expression under different conditions .

The presence of mobile genetic elements, such as the novel filamentous prophage integrated into the C. taiwanensis STM 6018 genome, could potentially influence the expression stability of nearby genes including tpiA . The integration of such elements often occurs at specific sites like the dif (deletion induced filamentation) site, which can affect the local genomic architecture and potentially influence gene expression .

Methodological approaches to investigate genetic context include:

• Comparative genomic analysis across Cupriavidus species to identify conserved synteny

• Transcriptomic analysis under various conditions to identify co-regulated genes

• Promoter mapping and analysis to identify regulatory elements

• Chromosome conformation capture techniques to identify long-range genomic interactions

What structural and functional characteristics make C. taiwanensis TpiA a potential target for specific inhibition?

Several characteristics of C. taiwanensis TpiA make it a promising target for specific inhibition:

TPIs from various organisms display structural differences that can be exploited for selective targeting. For example, TPIs from parasitic flatworms harbor a three-amino acid motif (SXD/E) not present in TPIs from non-parasitic flatworms or TPIs from hosts, making this region a putative target for drug design .

The active site of TPI consists of three invariable catalytic residues (Lys, His, and Glu) that are essential for enzymatic function . Compounds targeting these residues or nearby regions could specifically inhibit TpiA activity. Additionally, the dimeric nature of TpiA presents opportunities for developing compounds that disrupt protein-protein interactions at the dimer interface, potentially inactivating the enzyme .

Research in P. aeruginosa has identified TpiA as a key metabolic enzyme affecting bacterial virulence and antibiotic resistance, suggesting that targeting this enzyme could be a viable strategy for controlling bacterial growth and pathogenicity .

How can researchers design experiments to investigate the role of TpiA in C. taiwanensis stress response and adaptation?

To investigate the role of TpiA in C. taiwanensis stress response and adaptation, researchers should consider the following experimental design framework:

1. Genetic Manipulation Approaches:

• CRISPR-Cas9 mediated gene editing to create tpiA knockout/knockdown mutants

• Construction of tpiA overexpression strains

• Site-directed mutagenesis to modify specific functional domains

2. Stress Response Analysis Protocol:

• Subject wild-type and mutant strains to various stressors (oxidative, osmotic, temperature, pH)

• Measure growth rates, survival, and metabolic activity under stress conditions

• Analyze tpiA expression using qRT-PCR under different stress conditions

• Perform RNA-seq to identify genes co-regulated with tpiA during stress

3. Metabolic Flexibility Assessment:

• Growth profiling on different carbon sources

• 13C metabolic flux analysis under different growth conditions

• Metabolomics to identify changes in metabolite pools in response to stress

4. Structural Analysis in Stress Conditions:

• Thermal shift assays to assess protein stability under different conditions

• Hydrogen-deuterium exchange mass spectrometry to analyze structural dynamics

• Enzyme kinetics at different temperatures, pH values, and in the presence of oxidative agents

By implementing this comprehensive experimental framework, researchers can elucidate the specific roles of TpiA in C. taiwanensis stress response and adaptive mechanisms.

What comparative analyses of TpiA across Cupriavidus species might reveal about evolutionary conservation and specialization?

Comparative analyses of TpiA across Cupriavidus species can provide valuable insights into evolutionary conservation and functional specialization:

Phylogenetic analysis of C. taiwanensis STM 6018 shows that it is closely related to C. taiwanensis LMG 19424T, C. nantongensis X1T, C. alkaliphilus ASC-732T, and the rhizobial strain "C. neocaledonicus" STM 6070 . Comparative analysis of TpiA sequences across these species could reveal conservation patterns and species-specific adaptations.

Recommended Analytical Approaches:

• Multiple sequence alignment of TpiA sequences from different Cupriavidus species

• Identification of conserved catalytic residues and variable regions

• Analysis of selection pressure on different domains using dN/dS ratios

• Homology modeling to predict structural differences

• Correlation of sequence/structural differences with ecological niches

The study of TpiA across Cupriavidus species that occupy different ecological niches (such as free-living versus symbiotic species) could reveal adaptations related to lifestyle. For example, C. taiwanensis forms symbiotic relationships with Mimosa species , while other Cupriavidus species may have different metabolic priorities.