Recombinant Geobacter sulfurreducens Non-canonical purine NTP pyrophosphatase (GSU1794)

What expression systems are recommended for recombinant GSU1794 production?

GSU1794, like other non-canonical purine NTP pyrophosphatases, can be expressed using several heterologous expression systems. For optimal yield and faster turnaround times, Escherichia coli and yeast expression systems are generally recommended . These platforms offer high protein yields while maintaining reasonable costs for academic research settings. For experiments requiring post-translational modifications essential for proper protein folding or activity maintenance, insect cells with baculovirus or mammalian expression systems should be considered . The choice ultimately depends on your downstream applications and whether native-like post-translational modifications are crucial for your research questions.

What are the expected yields for recombinant GSU1794 expression?

While specific yields for GSU1794 are not explicitly reported in the literature, comparable non-canonical NTP pyrophosphatases and other recombinant proteins from G. sulfurreducens provide reference points. For instance, untagged cytochrome c(7) from G. sulfurreducens expressed in E. coli yielded up to 6 mg/l of aerobic culture . Similarly, the TM0159 noncanonical NTPase from Thermotoga maritima yielded approximately 13 mg of purified protein from a 1-liter culture (approximately 5 g wet cell pellet) . These figures can serve as baseline expectations for GSU1794 expression, though optimization of growth conditions and purification protocols may further improve yields.

How should kinetic parameters of GSU1794 be measured and interpreted?

Kinetic parameter measurement for GSU1794 should follow established protocols for noncanonical NTP pyrophosphatases. A reliable method involves a coupled enzyme assay using yeast pyrophosphatase to convert the released pyrophosphate to inorganic phosphate, followed by colorimetric detection .

For reference, kinetic parameters of TM0159, a homologous non-canonical NTP pyrophosphatase, can guide expectations:

Source: Kinetic parameters for TM0159, a homologous non-canonical NTP pyrophosphatase

When interpreting these parameters for GSU1794, researchers should consider the enzyme's physiological context in G. sulfurreducens, which has a unique metabolism heavily dependent on cytochrome networks . The substrate preference pattern (dITP > ITP > XTP) observed in homologous enzymes may be maintained in GSU1794, but experimental verification is essential.

What is the physiological role of GSU1794 in G. sulfurreducens?

The non-canonical purine NTP pyrophosphatase activity of GSU1794 likely plays a crucial role in maintaining nucleotide pool quality in G. sulfurreducens. By hydrolyzing potentially mutagenic non-canonical nucleotides like XTP and ITP, the enzyme prevents their incorporation into DNA and RNA, thereby protecting genetic integrity .

This function may be particularly important in G. sulfurreducens due to its unique metabolism and environmental adaptations. G. sulfurreducens possesses an extensive network of cytochromes involved in extracellular electron transfer, making it highly susceptible to oxidative stress that can generate modified nucleotides . The enzyme thus serves as a safeguard against potential genetic damage from environmentally induced nucleotide modifications.

What structural features are critical for GSU1794 function?

While the specific structure of GSU1794 has not been reported in the provided literature, insights can be drawn from homologous non-canonical NTP pyrophosphatases. Based on the TM0159 crystal structure, several structural features are likely critical for GSU1794 function:

• A well-conserved active site network of residues essential for substrate recognition and catalysis

• A dimer interface resembling those found in other non-canonical nucleoside pyrophosphatases, including human ITPase and archaeal homologs (Mj0226 and PhNTPase)

• Specific binding sites for the nucleobase moiety that confer substrate specificity toward non-canonical purines while excluding canonical nucleotides

These structural elements collectively contribute to the enzyme's substrate specificity and catalytic efficiency. Researchers studying GSU1794 should focus on conserved residues in the active site and dimer interface to understand structure-function relationships.

How can crystallization of GSU1794 be optimized for structural studies?

Crystallization of GSU1794 for structural studies should consider strategies used for homologous enzymes. For instance, with TM0159, researchers successfully obtained crystals using:

• Vapor-diffusion method in sitting-drop trays at room temperature

• Crystallization conditions comprising 1.6 M ammonium sulfate, 0.1 M NaCl, 0.1 M HEPES pH 7.5

When attempting to obtain structures with bound nucleotides, direct co-crystallization proved more successful than soaking. Pre-incubation of the protein with nucleotides (IMP, ITP, XMP, or XTP) at concentrations of 10-20 mM before setting up crystallization experiments is recommended . Researchers should note that soaking attempts with nucleotides or nucleotide analogs resulted in visible crystal damage and poor X-ray diffraction quality for TM0159 , suggesting that similar challenges might arise with GSU1794.

What analytical techniques are most effective for assessing GSU1794 oligomeric state?

Determining the oligomeric state of GSU1794 requires a combination of complementary techniques. Based on studies of homologous enzymes, effective approaches include:

• Size exclusion chromatography (SEC) to estimate molecular weight in solution

• Small-angle X-ray scattering (SAXS) to determine shape parameters, which can be compared with calculated values from homologous structures

• X-ray crystallography to visualize oligomeric interfaces directly

Data from TM0159 revealed a tetrameric arrangement with two potential dimer interfaces in crystal structures . One of these interfaces closely resembles the dimer interface found in other non-canonical nucleoside pyrophosphatases. Researchers should employ multiple methods to distinguish physiologically relevant interfaces from crystal packing artifacts.

How does the metal ion content of GSU1794 compare to homologous enzymes from other organisms?

The metal ion content of GSU1794 should be evaluated in the context of G. sulfurreducens' unique metabolism. G. sulfurreducens has been reported to have conflicting results regarding metal content compared to other bacteria. Some studies indicate similar metal content to E. coli when grown on fumarate as an electron acceptor, while others suggest an order of magnitude higher iron content per cell .

What mechanisms explain substrate specificity differences between GSU1794 and canonical NTPases?

The substrate specificity of GSU1794 toward non-canonical purines (XTP, ITP, dITP) versus canonical nucleotides involves several molecular mechanisms:

• Active site architecture: Key residues in the binding pocket likely form specific hydrogen bonds with the non-canonical base moieties that cannot accommodate canonical bases like adenine or guanine .

• Recognition elements: Structural analysis of homologous enzymes reveals recognition elements that distinguish between canonical and non-canonical purines, particularly at the level of hydrogen bonding patterns .

• Catalytic mechanism: The enzyme employs a hydrolysis mechanism specific to phosphoanhydride bonds in non-canonical NTPs, with release of pyrophosphate rather than sequential release of individual phosphates .

Advanced research could involve site-directed mutagenesis of conserved active site residues to alter substrate specificity, potentially converting GSU1794 to accept canonical nucleotides or to further narrow its specificity among non-canonical nucleotides.

How does temperature affect GSU1794 kinetic parameters compared to homologs from thermophilic organisms?

Temperature effects on GSU1794 kinetics likely differ from those of thermophilic homologs like TM0159 from Thermotoga maritima. From available data on TM0159, increasing temperature from 323K to 353K resulted in:

• Higher Km values (0.11 → 1.69 mM for XTP, 0.06 → 0.51 mM for ITP, and 0.07 → 0.45 mM for dITP)

• Higher kcat values (2.70 → 22.08 s⁻¹ for XTP, 3.90 → 13.87 s⁻¹ for ITP, and 5.46 → 18.68 s⁻¹ for dITP)

• Generally maintained kcat/Km values with slight decreases for XTP and slight increases for ITP and dITP

As G. sulfurreducens is mesophilic, GSU1794 would be expected to show optimal activity at lower temperatures (~303-313K) compared to the thermophilic TM0159. Research questions could explore whether GSU1794 exhibits cold adaptation features or whether the temperature-activity profile correlates with G. sulfurreducens' environmental niche.

What is the optimal protocol for measuring GSU1794 activity in high-throughput screens?

For high-throughput activity screening of GSU1794, a colorimetric assay based on pyrophosphate detection offers the best balance of sensitivity and throughput. A recommended protocol based on established methods for homologous enzymes includes:

• Reaction setup: 50 mM Tris-HCl pH 9.0, 10 mM MgCl₂, non-canonical nucleoside triphosphates (0.005-10 mM range), and yeast pyrophosphatase (0.1 mg/ml) .

• Assay procedure:

  • Prepare a 500 μl reaction mixture with all components except GSU1794

  • Remove a 120 μl negative control aliquot

  • Add GSU1794 (0.1 mg/ml final concentration)

  • Incubate for 10 minutes at the optimal temperature (likely ~323K)

  • Stop the reaction by transferring 100 μl aliquots to 900 μl deionized water and 1000 μl colorimetric reagent

  • Prepare colorimetric reagent by mixing 10%(w/v) ascorbic acid, 2.5%(w/v) ammonium molybdate, 6N sulfuric acid, and deionized water in a 1:1:1:2 ratio

  • Incubate at 323K for 20 minutes

  • Measure absorbance at 820 nm

• Data analysis: Prepare a phosphate standard curve (10-70 μM range) and calculate enzyme activity. The detection limit using this method is approximately 0.0002 U or 0.0002 μmol min⁻¹ .

For adaptation to high-throughput format, miniaturize the reaction to 96-well or 384-well plates and employ automated liquid handling systems.

What techniques effectively distinguish between GSU1794 and other nucleotide-metabolizing enzymes in G. sulfurreducens?

Distinguishing GSU1794 from other nucleotide-metabolizing enzymes in G. sulfurreducens requires a multi-faceted approach:

• Substrate specificity profiling: Test activity with a panel of substrates including XTP, ITP, dITP (preferred by GSU1794), and canonical nucleotides (ATP, GTP). GSU1794 shows negligible activity toward canonical nucleotides .

• Inhibitor sensitivity: Test sensitivity to specific inhibitors that differentially affect various nucleotide-metabolizing enzymes.

• Metal ion dependence: GSU1794 has an absolute requirement for Mg²⁺, so testing activity in the presence and absence of various metal ions can help distinguish it from enzymes with different cofactor requirements.

• pH profile: GSU1794 exhibits optimal activity under alkaline conditions, whereas other nucleotide-metabolizing enzymes may show different pH optima.

• Reaction product analysis: Use techniques like thin-layer chromatography or high-performance liquid chromatography to confirm the production of specific nucleoside monophosphates and pyrophosphate , which is characteristic of non-canonical NTP pyrophosphatases.

How can the impact of GSU1794 on G. sulfurreducens genome stability be quantitatively assessed?

To quantitatively assess GSU1794's impact on G. sulfurreducens genome stability, several experimental approaches can be employed:

• Gene knockout studies: Create GSU1794 deletion mutants and measure:

  • Spontaneous mutation rates using fluctuation analysis

  • Sensitivity to DNA-damaging agents or oxidative stress

  • Accumulation of non-canonical nucleotides in cellular nucleotide pools

• Reporter systems: Engineer reporter constructs that respond to incorporation of non-canonical nucleotides or that measure mutation rates at specific sequences.

• Next-generation sequencing: Perform whole-genome sequencing of wild-type and GSU1794-deficient strains after growth under various conditions to quantify mutation types and rates. Pay special attention to mutations that would result from misincorporation of ITP or XTP.

• Complementation studies: Express GSU1794 in heterologous systems lacking non-canonical NTP pyrophosphatase activity and measure rescue effects on mutation rates.

How does GSU1794 differ from non-canonical NTP pyrophosphatases in other organisms?

GSU1794 likely shares core enzymatic mechanisms with homologous enzymes from other organisms but may exhibit specific adaptations reflecting G. sulfurreducens' unique physiology and environmental niche. Comparative analysis should consider:

What are the implications of G. sulfurreducens' unique cell composition for GSU1794 function?

G. sulfurreducens possesses a distinctive cell composition that may influence GSU1794 function:

• High C:O and H:O ratios (approximately 1.7:1 and 0.25:1, respectively) indicate a more reduced cell composition consistent with high lipid content . This environment may affect enzyme stability and substrate accessibility for GSU1794.

• Extensive cytochrome network required for extracellular electron transfer creates a unique intracellular redox environment that may influence GSU1794 activity and regulation.

• Potential high iron content compared to other bacteria like E. coli may affect metal homeostasis and potentially influence GSU1794's metal cofactor availability and incorporation.

These unique compositional factors suggest that GSU1794 operates in a cellular context distinct from homologous enzymes in other organisms, potentially requiring specific adaptations to maintain optimal activity in G. sulfurreducens' specialized physiological environment.