Recombinant Solanum habrochaites Fructokinase-2 (FRK2)

What is Fructokinase-2 (FRK2) and what role does it play in Solanum habrochaites?

Fructokinase-2 (FRK2) is a key enzyme in carbohydrate metabolism that catalyzes the phosphorylation of fructose to fructose-6-phosphate, an essential step in both glycolysis and sucrose metabolism. In Solanum habrochaites, FRK2 plays a particularly important role in determining fruit sugar composition. The enzyme is involved in an epistatic interaction with the frg allele, which results in a higher ratio of fructose to glucose in the fruit, a trait desirable for sweetness enhancement as fructose is sweeter than glucose . This unique sugar profile distinguishes S. habrochaites from cultivated tomato varieties (Solanum lycopersicum), making it a valuable genetic resource for breeding programs aimed at improving fruit quality traits.

How does S. habrochaites FRK2 differ from FRK2 in cultivated tomato (S. lycopersicum)?

S. habrochaites FRK2 differs from its S. lycopersicum counterpart in several key aspects:

• Structural differences: The protein sequences show specific amino acid variations that may affect substrate binding and catalytic efficiency.

• Expression patterns: S. habrochaites FRK2 shows distinct temporal and spatial expression patterns compared to cultivated tomato.

• Functional properties: The wild species enzyme exhibits unique kinetic properties, particularly in its interaction with the frg allele, contributing to the characteristic higher fructose-to-glucose ratio observed in S. habrochaites fruits .

• Regulatory mechanisms: Different transcriptional and post-translational regulation mechanisms likely exist between the two species, affecting enzyme activity and stability.

These differences contribute to the distinct carbohydrate metabolism observed in S. habrochaites, particularly its ability to maintain a favorable fructose-to-glucose ratio, which has implications for both stress tolerance and fruit sweetness.

What are the primary substrates and products of FRK2 enzymatic activity?

FRK2 primarily catalyzes the ATP-dependent phosphorylation of fructose:

Fructose + ATP → Fructose-6-phosphate + ADP

The main substrates for the reaction are:

• Fructose: The primary sugar substrate

• ATP: The phosphoryl donor

The products of the reaction are:

• Fructose-6-phosphate: A key intermediate in both glycolysis and sucrose metabolism

• ADP: The nucleotide product after phosphoryl transfer

FRK2 shows high specificity for fructose as its sugar substrate, with minimal activity toward other hexoses. This substrate specificity is critical for its role in directing carbon flow from fructose into primary metabolism. In S. habrochaites, this enzymatic activity contributes to the distinctive sugar profile observed in fruits, particularly when interacting with the frg allele to produce a higher fructose-to-glucose ratio .

How does the epistatic interaction between FRK2 and the frg allele affect sugar metabolism in S. habrochaites?

The epistatic interaction between FRK2 and the frg allele in S. habrochaites creates a complex regulatory network that significantly impacts sugar metabolism. This interaction leads to a higher fructose-to-glucose ratio compared to cultivated tomato varieties . The molecular mechanism behind this interaction involves:

• Altered enzyme kinetics: The FRK2 enzyme in S. habrochaites exhibits different kinetic properties when the frg allele is present, potentially affecting its affinity for fructose or its catalytic efficiency.

• Metabolic flux redirection: The interaction alters carbon flux through glycolysis and associated pathways, favoring fructose accumulation over glucose.

• Regulatory feedback loops: The interaction likely influences expression levels of other sugar metabolism genes through complex feedback mechanisms.

• Developmental timing: The epistatic effects show temporal specificity, becoming more pronounced during fruit ripening when sugar accumulation is highest.

Previous attempts to engineer high fructose content by manipulating fructokinase activity alone have been unsuccessful , highlighting the importance of understanding this epistatic interaction for successful breeding programs. The molecular details of how the frg allele modifies FRK2 function remain incompletely characterized, representing an important area for future research.

What structural features of recombinant S. habrochaites FRK2 contribute to its unique enzymatic properties?

The unique enzymatic properties of recombinant S. habrochaites FRK2 can be attributed to several structural features:

These structural attributes collectively contribute to the enzyme's ability to participate in the epistatic interaction with the frg allele, resulting in the characteristic higher fructose-to-glucose ratio observed in S. habrochaites fruits . Detailed structural studies using X-ray crystallography or cryo-electron microscopy would provide valuable insights into these features.

How does environmental stress affect FRK2 expression and activity in S. habrochaites?

Environmental stress significantly modulates both the expression and activity of FRK2 in S. habrochaites, contributing to the species' notable stress tolerance. Under various stress conditions, the following patterns have been observed:

• Drought stress: FRK2 expression increases under moderate drought conditions, potentially contributing to osmotic adjustment through altered sugar metabolism. This may relate to the QTL identified for water stress tolerance in S. habrochaites, which could involve sugar metabolism genes .

• Temperature stress:

  • Cold stress: FRK2 shows enhanced expression at low temperatures, potentially contributing to cold acclimation

  • Heat stress: The enzyme exhibits altered kinetic properties at high temperatures, with apparent adaptation to maintain activity

• Pathogen stress: During infection with pathogens like Pseudomonas syringae, sugar metabolism pathways including those involving FRK2 undergo significant reconfiguration, potentially contributing to defense responses .

• Post-translational regulation: Environmental stresses trigger changes in phosphorylation status and protein-protein interactions that modify FRK2 activity without necessarily altering transcript levels.

These stress-responsive properties of FRK2 likely contribute to S. habrochaites' remarkable ability to maintain metabolic homeostasis under adverse conditions, including its documented tolerance to root chilling . The stress-responsive characteristics of FRK2 make it a promising candidate gene for improving stress tolerance in cultivated tomato varieties.

What are the optimal conditions for expressing recombinant S. habrochaites FRK2 in bacterial systems?

Optimal conditions for expressing recombinant S. habrochaites FRK2 in bacterial systems include:

Expression System Parameters:

Optimization Strategies:

• Codon optimization: Adapting the S. habrochaites sequence to E. coli codon usage can significantly improve expression levels

• Fusion partners: Addition of fusion tags like MBP or SUMO can enhance solubility

• Chaperone co-expression: Co-expressing molecular chaperones (GroEL/GroES) can improve folding

• Supplementation with 1% glucose in pre-induction media can reduce basal expression

This approach has been successfully adapted from methods used for expressing similar plant enzymes involved in specialized metabolism, such as the methylketone synthases from S. habrochaites . Proper optimization of these conditions is critical for obtaining enzymatically active FRK2 for subsequent biochemical and structural characterization.

What techniques are most effective for purifying recombinant S. habrochaites FRK2 while maintaining enzymatic activity?

Purification of recombinant S. habrochaites FRK2 requires careful technique selection to preserve enzymatic activity:

Multi-step Purification Protocol:

• Initial Lysis and Clarification:

  • Buffer composition: 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF

  • Sonication: 6 cycles of 10s on/20s off at 40% amplitude

  • Clarification: Centrifugation at 20,000 × g for 30 minutes at 4°C

• Immobilized Metal Affinity Chromatography (IMAC):

  • Resin: Ni-NTA agarose

  • Binding: 20 mM imidazole in base buffer

  • Washing: Stepwise with 50 mM and 80 mM imidazole

  • Elution: 250 mM imidazole in base buffer

  • Critical stabilizers: 10% glycerol and 1 mM DTT throughout

• Size Exclusion Chromatography:

  • Column: Superdex 200 10/300 GL

  • Buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.5 mM DTT

  • Flow rate: 0.5 mL/min

• Activity Preservation Factors:

  • Temperature control: Maintain 4°C throughout purification

  • Enzyme stabilizers: 10% glycerol prevents aggregation

  • Reducing agents: 1 mM DTT protects critical cysteine residues

  • Metal ions: Addition of 0.1 mM MgCl₂ helps maintain structural integrity

  • Protease inhibitor cocktail: Essential during initial lysis steps

Purification Assessment:

This purification approach has been successfully adapted from methods used for similar metabolic enzymes from wild tomato species , with specific modifications to accommodate the unique properties of FRK2.

How can enzymatic activity of recombinant S. habrochaites FRK2 be measured accurately?

Accurate measurement of recombinant S. habrochaites FRK2 enzymatic activity requires sensitive and specific assays: