Recombinant Desulfovibrio vulgaris Xanthine phosphoribosyltransferase (gpt)

What genetic systems are most effective for expressing recombinant Xanthine phosphoribosyltransferase in D. vulgaris?

D. vulgaris presents unique challenges for genetic manipulation, but recent advances have significantly improved transformation efficiency. For optimal expression of Xanthine phosphoribosyltransferase, the markerless genetic exchange system utilizing uracil phosphoribosyltransferase (upp) as a counterselectable marker has proven highly effective . This system allows for creation of clean deletions and insertions without retaining antibiotic resistance markers.

The transformation efficiency can be dramatically improved (100-1000 fold) by using the JW7035 strain that has a deletion of the hsdR gene encoding a type I restriction endonuclease . When using electroporation for transformation, plasmids containing the endogenous SRB cryptic plasmid pBG1 yield better results for expression systems.

What are the optimal induction conditions for maximal expression of functional recombinant Xanthine phosphoribosyltransferase in D. vulgaris?

The expression of recombinant Xanthine phosphoribosyltransferase in D. vulgaris is optimized using promoters that function efficiently in sulfate-reducing bacteria. The aph(3′)-II promoter (from the kanamycin resistance gene in Tn5) has demonstrated strong constitutive expression for recombinant proteins in D. vulgaris . For inducible expression, the following parameters should be considered:

How does the purification strategy for recombinant Xanthine phosphoribosyltransferase from D. vulgaris differ from other bacterial expression systems?

Purification of recombinant Xanthine phosphoribosyltransferase from D. vulgaris requires special considerations due to the anaerobic nature of this organism and potential structural differences compared to phosphoribosyltransferases from other organisms. Based on methods used for similar enzymes, a recommended purification protocol includes:

• Cell lysis under anaerobic conditions using a buffer containing reducing agents to prevent oxidation of sensitive thiol groups

• Initial capture using immobilized metal affinity chromatography (IMAC) if a His-tag is employed

• Affinity chromatography using GTP-agarose, which has proven effective for purifying recombinant phosphoribosyltransferases to homogeneity, similar to the approach used for T. brucei HGPRT

• Size exclusion chromatography as a polishing step to remove aggregates and obtain the native oligomeric state

This approach differs from standard bacterial systems primarily in the anaerobic requirements and the need to maintain reducing conditions throughout the purification process.

What substrates does recombinant D. vulgaris Xanthine phosphoribosyltransferase recognize, and how does its substrate specificity compare to related enzymes?

Enzymatic assays to determine substrate specificity should include a range of potential purine substrates and utilize methods such as spectrophotometric monitoring of product formation or HPLC-based detection of nucleotide products.

How does the kinetic mechanism of D. vulgaris Xanthine phosphoribosyltransferase compare with other phosphoribosyltransferases?

Many phosphoribosyltransferases, including the P. falciparum HGXPRT, follow an ordered sequential bi-bi kinetic mechanism where PRPP·Mg²⁺ binds first, followed by the purine base . The kinetic mechanism can be investigated using:

• Initial velocity studies with varied concentrations of both substrates

• Product inhibition patterns

• Dead-end inhibitor studies

• Isotope exchange at equilibrium

Some phosphoribosyltransferases demonstrate activation mechanisms involving conformational changes. For example, P. falciparum HGXPRT exhibits a slow ligand-induced conformational switch that increases its catalytic rate, where pre-incubation with substrate/products switches the enzyme from a low activity to a high activity state . This activation involves:

• Reduction in Km for PRPP·Mg²⁺ by up to 10-fold

• Elimination of lag phase in reaction progress curves

• Ligand-mediated oligomerization

Researchers should investigate whether D. vulgaris Xanthine phosphoribosyltransferase exhibits similar activation phenomena by monitoring reaction progress under varying pre-incubation conditions.

What are the optimal methodologies for measuring enzyme activity and inhibition of recombinant D. vulgaris Xanthine phosphoribosyltransferase?

Multiple complementary approaches can be employed to accurately characterize the activity and inhibition of D. vulgaris Xanthine phosphoribosyltransferase:

• Spectrophotometric continuous assays: Monitor the change in absorbance at 257 nm (xanthine) to 290 nm (XMP)

• Radiochemical assays: Using ¹⁴C-labeled substrates to measure product formation with high sensitivity

• HPLC-based assays: Quantify product formation by separating nucleotides chromatographically

• Coupled enzyme assays: Link product formation to a secondary enzymatic reaction with a detectable output

For inhibition studies, researchers should consider:

• Determining IC₅₀ values across a range of substrate concentrations

• Establishing inhibition mechanisms (competitive, non-competitive, uncompetitive, mixed)

• Evaluating time-dependent inhibition to identify slow-binding inhibitors

• Using thermal shift assays to assess inhibitor binding through protein stabilization

What structural elements contribute to substrate specificity in D. vulgaris Xanthine phosphoribosyltransferase, and how can they be modified for altered functionality?

Phosphoribosyltransferases share conserved structural elements while maintaining substrate specificity through variations in their binding pockets. Key structural elements likely include:

• A conserved PPi binding site that often contains a Leu-Lys dipeptide which facilitates PRPP·Mg²⁺ binding through isomerization from trans to cis conformation

• A purine base binding pocket with residues determining specificity for xanthine versus other purines

• Metal binding sites for catalytic Mg²⁺ ions

For modifying substrate specificity, site-directed mutagenesis approaches should target:

• Residues lining the purine binding pocket

• Loops that undergo conformational changes upon substrate binding

• Interface residues involved in oligomerization, which may impact catalytic activity

Energy calculation approaches similar to those used with P. falciparum HGXPRT, such as well-tempered metadynamics techniques, can help predict the impact of mutations on conformational stability and substrate binding .

How can molecular dynamics simulations inform the design of high-affinity inhibitors targeting D. vulgaris Xanthine phosphoribosyltransferase?

Molecular dynamics (MD) simulations can provide critical insights for rational inhibitor design by:

• Identifying transient binding pockets not visible in static crystal structures

• Characterizing the conformational energy landscape, particularly for elements like the Leu-Lys dipeptide that undergoes cis-trans isomerization

• Predicting water networks important for ligand binding

• Calculating binding free energies for potential inhibitors

Researchers should focus on:

• Running long timescale (>100 ns) simulations to capture relevant conformational changes

• Employing enhanced sampling techniques like metadynamics to overcome energy barriers

• Including explicit solvent models to accurately represent the enzyme's native environment

• Validating computational predictions with experimental binding and kinetic data

For the Leu-Lys dipeptide specifically, free energy calculations can determine whether ligand-free enzyme is more stable with this element in trans or cis conformation, similar to what has been observed for P. falciparum HGXPRT .

What genetic techniques are most effective for creating knockout or conditional mutants of Xanthine phosphoribosyltransferase in D. vulgaris for physiological studies?

Creating knockout or conditional mutants in D. vulgaris has been significantly advanced by the development of the markerless genetic exchange system using the upp gene (encoding uracil phosphoribosyltransferase) as a counterselectable marker . This two-step recombination approach allows for:

• Generation of clean deletions without retaining antibiotic resistance markers

• Sequential deletion of multiple genes without accumulating selection markers

• Introduction of conditional expression systems

The practical implementation involves:

• Construction of a suicide plasmid containing upstream and downstream flanking regions of the target gene

• Expression of wild-type upp gene from the aph(3′)-II promoter

• Selection of integration using resistance to an appropriate antibiotic

• Counter-selection of excision events using 5-fluorouracil (5-FU) resistance

Transformation efficiency can be significantly improved by using the JW7035 strain that lacks the hsdR gene encoding a type I restriction endonuclease, which increases transformation rates by 100-1000 fold compared to wild-type strains .

How does deletion of Xanthine phosphoribosyltransferase affect the metabolic profile and stress response of D. vulgaris?

Xanthine phosphoribosyltransferase plays a role in purine salvage pathways, and its deletion would likely impact nucleotide metabolism and potentially stress responses in D. vulgaris. Based on studies of related enzymes in other organisms, researchers should investigate:

• Changes in purine nucleotide pools using targeted metabolomics

• Alterations in growth rates under different nutrient conditions

• Modifications in stress response pathways, particularly those involving nucleotide-dependent signaling

• Potential cross-talk with sulfate reduction pathways that are central to D. vulgaris metabolism

D. vulgaris is known to cause gut inflammation and aggravate DSS-induced colitis , and studying the role of Xanthine phosphoribosyltransferase in this context could provide insights into bacterial factors contributing to inflammatory responses.

How can recombinant D. vulgaris Xanthine phosphoribosyltransferase be utilized as a selection marker for genetic manipulation in anaerobic bacteria?

Phosphoribosyltransferases have been successfully employed as selection markers in various genetic systems. For D. vulgaris and other anaerobic bacteria, Xanthine phosphoribosyltransferase could be developed as a selection marker by:

• Creating a host strain with deleted native Xanthine phosphoribosyltransferase gene

• Developing a complementation system where the gene is expressed from a plasmid

• Establishing selection conditions where purine salvage through this enzyme provides a growth advantage

This approach offers several advantages:

• Provides an alternative to antibiotic resistance markers

• Enables selection under strictly anaerobic conditions

• Allows for positive and negative selection strategies

• Can be combined with other marker systems for complex genetic manipulations

The development would require establishing appropriate selection media that force reliance on the purine salvage pathway facilitated by this enzyme.

What are the most effective approaches for studying protein-protein interactions involving D. vulgaris Xanthine phosphoribosyltransferase in its native cellular context?

Studying protein-protein interactions in anaerobic bacteria presents unique challenges. For D. vulgaris Xanthine phosphoribosyltransferase, the following approaches are recommended:

• In vivo crosslinking: Using membrane-permeable crosslinkers followed by affinity purification and mass spectrometry identification of interaction partners

• Split-protein complementation assays: Adapted for use in anaerobic bacteria

• Co-immunoprecipitation with antibodies: Against native Xanthine phosphoribosyltransferase or epitope-tagged versions

• Bacterial two-hybrid systems: Modified for expression in D. vulgaris or in surrogate hosts

These approaches should be combined with confirmatory biochemical assays to validate interactions and assess their functional significance. Researchers should consider the potential impact of the anaerobic environment on these interactions, particularly for redox-sensitive proteins.

How does D. vulgaris Xanthine phosphoribosyltransferase activity contribute to bacterial fitness during host colonization and infection?

D. vulgaris has been shown to cause gut inflammation and aggravate DSS-induced colitis , making the role of its metabolic enzymes in host colonization a relevant research question. To investigate the contribution of Xanthine phosphoribosyltransferase to this process:

• Compare wild-type and Xanthine phosphoribosyltransferase knockout strains in:

  • Colonization efficiency in animal models

  • Competitive fitness assays in gut environments

  • Survival under host-imposed stresses (nutrient limitation, immune factors)

• Analyze transcriptional responses in both the bacterium and host during infection using RNA-seq approaches

• Investigate whether Xanthine phosphoribosyltransferase or its metabolic products directly influence host inflammatory responses by:

  • Measuring cytokine production in response to bacterial extracts

  • Assessing changes in gut epithelial barrier function

  • Evaluating alterations in host immune cell recruitment and activation

Given that D. vulgaris flagellin has been shown to exacerbate colitis through interaction with LRRC19 and subsequent pro-inflammatory cytokine secretion , researchers should explore potential connections between purine metabolism and virulence factor expression.

How does D. vulgaris Xanthine phosphoribosyltransferase compare structurally and functionally with homologous enzymes from other sulfate-reducing bacteria?

A comprehensive comparative analysis should include:

• Sequence alignment analysis to identify conserved motifs and divergent regions

• Homology modeling based on crystal structures of related enzymes

• Functional comparison through heterologous expression and enzymatic characterization

• Phylogenetic analysis to understand evolutionary relationships

The comparative analysis should specifically examine:

• Substrate specificity determinants across different bacterial lineages

• Conservation of catalytic residues and binding site architecture

• Structural adaptations related to the anaerobic lifestyle of sulfate-reducing bacteria

• Potential horizontal gene transfer events that shaped the evolution of these enzymes

What evolutionary adaptations in D. vulgaris Xanthine phosphoribosyltransferase reflect the specialized metabolic requirements of sulfate-reducing bacteria?

Sulfate-reducing bacteria like D. vulgaris have specialized metabolic requirements that may be reflected in adaptations of their purine salvage enzymes. Researchers should investigate:

• Potential adaptations to function optimally under reducing conditions characteristic of sulfate-reducing bacteria

• Modifications that might coordinate purine metabolism with sulfate reduction pathways

• Structural features that enhance stability in environments with high sulfide concentrations

• Regulatory mechanisms that integrate purine salvage with central metabolic processes in anaerobic energy conservation

These evolutionary adaptations can be identified through:

• Ancestral sequence reconstruction and resurrection

• Experimental evolution under varying selective pressures

• Comparative genomics across sulfate-reducing bacteria with different metabolic capabilities

• Analysis of natural variation in enzyme properties across D. vulgaris strains from different environments

What are the most common challenges in expressing and purifying active recombinant D. vulgaris Xanthine phosphoribosyltransferase, and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant proteins from anaerobic bacteria like D. vulgaris:

For expression in D. vulgaris specifically, using the markerless genetic exchange system with the upp gene as a counterselectable marker provides significant advantages for generating expression strains with stable integration .

How can researchers troubleshoot inconsistent kinetic data when characterizing D. vulgaris Xanthine phosphoribosyltransferase?

Inconsistent kinetic data often stems from several factors that can be methodically addressed:

• Enzyme stability issues:

  • Monitor enzyme activity over time under assay conditions

  • Determine and eliminate factors causing time-dependent inactivation

  • Test different storage conditions and additives to maintain activity

• Assay interference:

  • Validate assay methods with control enzymes of known activity

  • Test for interfering compounds in buffer components

  • Employ multiple orthogonal assay methods to cross-validate results

• Conformational heterogeneity:

  • Investigate potential activation mechanisms similar to P. falciparum HGXPRT

  • Test pre-incubation with substrates or products to achieve consistent activation state

  • Analyze oligomeric state by size exclusion chromatography or analytical ultracentrifugation

• Substrate quality:

  • Verify substrate purity by HPLC or mass spectrometry

  • Prepare fresh solutions of unstable substrates

  • Standardize substrate preparation protocols

When troubleshooting, researchers should systematically document conditions and results, and consider kinetic models that account for complex mechanisms like substrate activation, product inhibition, or cooperativity.