Recombinant Xylella fastidiosa Probable S-methyl-5'-thioinosine phosphorylase (mtnP)

What is the functional role of S-methyl-5'-thioinosine phosphorylase (mtnP) in Xylella fastidiosa metabolism?

S-methyl-5'-thioinosine phosphorylase (mtnP) is a critical enzyme in the methionine salvage pathway that catalyzes the phosphorolytic cleavage of S-methyl-5'-thioinosine to form hypoxanthine and S-methyl-5-thio-alpha-D-ribose 1-phosphate. The reaction can be represented as:

S-methyl-5'-thioinosine + phosphate → hypoxanthine + S-methyl-5-thio-alpha-D-ribose 1-phosphate

In bacteria like Xylella fastidiosa, this enzyme plays a vital role in recycling sulfur-containing metabolites and maintaining proper nucleotide pools. The methionine salvage pathway is particularly important for X. fastidiosa as a plant pathogen because it allows the bacterium to recycle sulfur-containing metabolites that may be limited in plant host environments. This pathway also contributes to the bacterium's ability to synthesize methionine, which is essential for protein synthesis and various cellular processes .

How does mtnP expression relate to X. fastidiosa's pathogenicity in plant hosts?

The expression of mtnP in Xylella fastidiosa is likely connected to its pathogenicity through several mechanisms. X. fastidiosa is a plant pathogen that causes serious diseases in various agricultural crops worldwide . The ability to efficiently recycle metabolites through pathways involving mtnP may provide competitive advantages when colonizing plant vascular systems.

X. fastidiosa demonstrates significant variability between strains regarding virulence on specific host plant species . This variability could be partly attributed to differences in metabolic efficiency, including variations in the methionine salvage pathway where mtnP functions. The ability to efficiently process and recycle sulfur-containing metabolites may influence the bacterium's fitness within specific plant hosts.

Additionally, natural competence and horizontal gene transfer occur frequently in X. fastidiosa and influence its evolution and adaptation to different environments . Variations in genes encoding metabolic enzymes like mtnP could potentially be transferred between strains, contributing to changes in host specificity or virulence.

What are the common methods for cloning and expressing recombinant mtnP from X. fastidiosa?

When expressing recombinant mtnP from X. fastidiosa, researchers typically employ several methodological approaches:

• Vector Selection: Expression vectors with appropriate promoters for bacterial expression (like pET system vectors) are commonly selected. The choice depends on whether constitutive or inducible expression is desired .

• Host Selection: While Escherichia coli is the most common expression host (particularly BL21(DE3) strain), expression levels can vary significantly based on the specific construct design .

• Codon Optimization: Given that X. fastidiosa has different codon usage patterns than E. coli, codon optimization of the mtnP gene may improve translation efficiency and expression levels in the heterologous host .

• Tag Selection and Placement: For purification purposes, affinity tags such as His6-tag are commonly added either at the N- or C-terminus. The location of the tag can significantly impact expression levels and protein solubility .

• Expression Optimization: Several parameters can be optimized:

  • IPTG concentration (typically 0.1-1 mM)

  • Induction temperature (often lower temperatures like 16-25°C improve solubility)

  • Induction duration (4-24 hours)

  • Culture media composition

How can N-terminal truncation strategies improve recombinant expression of X. fastidiosa mtnP?

Based on recent protein engineering studies, N-terminal truncation strategies can significantly enhance recombinant protein expression in E. coli systems. For optimizing X. fastidiosa mtnP expression, researchers should consider the following methodological approach:

• Structural and Phylogenetic Analysis: Begin by analyzing the crystal structure of mtnP and comparing it with orthologs from related species. Identify potential flexible or disordered regions at the N-terminus that might impede efficient expression .

• Multiple Truncation Design: Design several truncation constructs by removing different lengths of the N-terminal sequence. For example, if working with a full-length protein of 200 amino acids, create truncation constructs removing 15-20 amino acids at a time from the N-terminus (e.g., constructs starting at positions 215, 216, 217, 218) .

• Translation Initiation Rate (TIR) Analysis: Use computational algorithms to predict the TIR of each construct. Higher TIR values (e.g., >15,000 arbitrary units) generally correlate with better expression levels .

• Amino Acid Context Analysis: Examine the first few amino acids of each construct. The presence of lysine at the third position (as in M1-G2-K3-...) may enhance expression, while multiple successive proline residues can stall translation and reduce expression efficiency .

• Tag Placement Optimization: For N-terminal truncation constructs, C-terminal His6-tags often yield better results as they avoid interfering with the optimized N-terminus .

In a comparative study of similar enzymes, N-terminal truncation resulted in up to 28-fold increase in soluble protein yield (from 3-5 mg/L to 100-120 mg/L of culture), demonstrating the significant impact this strategy can have on expression efficiency .

What approaches can be used to characterize the substrate specificity of X. fastidiosa mtnP compared to orthologs from other bacterial species?

Characterizing substrate specificity of X. fastidiosa mtnP requires a multi-faceted approach:

• Enzyme Kinetics Analysis:

  • Measure Michaelis-Menten kinetics parameters (Km, kcat, kcat/Km) for the primary substrate (S-methyl-5'-thioinosine) and potential alternative substrates

  • Use spectrophotometric assays that monitor either substrate consumption or product formation

  • Include appropriate controls with known orthologs like the P. aeruginosa enzyme

• Comparative Substrate Panel Testing:

  • Test a panel of structurally related nucleosides including:

    • S-methyl-5'-thioinosine (primary substrate)

    • Inosine

    • 5'-methylthioadenosine

    • Other modified nucleosides

• Site-Directed Mutagenesis:

  • Identify putative substrate-binding residues based on structural information

  • Create single and multiple mutants of these residues

  • Characterize changes in substrate specificity for each mutant

• Structural Analysis:

  • Obtain crystal structures of the enzyme with bound substrates or substrate analogs

  • Perform molecular docking simulations with various substrates

  • Use molecular dynamics simulations to analyze substrate-enzyme interactions

• Phylogenetic Analysis:

  • Compare substrate preferences across mtnP orthologs from different bacterial species

  • Correlate amino acid differences in the binding pocket with substrate preference changes

  • Identify evolutionary patterns in substrate specificity

This comprehensive approach allows researchers to fully characterize the substrate specificity profile of X. fastidiosa mtnP and understand how it may differ from orthologs in other bacterial species like P. aeruginosa, where S-methyl-5'-thioinosine phosphorylase is known to participate in 5'-methylthioadenosine catabolism .

How might restriction-modification systems affect the genetic manipulation of mtnP in X. fastidiosa?

Genetic manipulation of mtnP in X. fastidiosa faces challenges due to the bacterium's complex restriction-modification (R-M) systems. A systematic approach to address these challenges includes:

• Identification of Active R-M Systems:

  • X. fastidiosa genomes contain several type I R-M systems that may restrict foreign DNA

  • These R-M systems are heterogeneous across X. fastidiosa strains, with different functional complements

  • Analysis of 129 X. fastidiosa genome assemblies identified three type I R-M systems conserved across all strains, plus an additional system in subspecies multiplex and pauca

• Methylation Pattern Analysis:

  • Characterize genomic DNA methylation patterns in the specific X. fastidiosa strain to be manipulated

  • Associate methylation patterns with type I R-M system allele profiles to predict recognition sites

  • 44 unique target recognition domains (TRDs) arranged in 50 unique hsdS alleles have been identified across X. fastidiosa strains

• Transformation Optimization Strategies:

  • Pre-methylate plasmid DNA using cell extracts from the target X. fastidiosa strain

  • Temporarily inhibit or delete type I R-M systems to increase transformation efficiency

  • Design constructs avoiding recognition sequences of the strain's active R-M systems

• Strain Selection:

  • Some X. fastidiosa strains contain inactivating mutations in their type I R-M systems

  • Select strains with fewer active R-M systems for initial genetic manipulations

  • Identify strains with frameshift mutations in hsdR, hsdM, or hsdS components

• Natural Competence Exploitation:

  • Utilize X. fastidiosa's natural competence for transformation with homologous DNA

  • Design DNA constructs with extensive homology to the target locus

  • Time transformations with the expression of competence genes

Understanding that type I R-M systems in X. fastidiosa undergo recombination and exchange of TRDs between specificity subunits (hsdS) is crucial, as this generates novel alleles with new target specificities . This recombination adds another layer of complexity when designing strategies for genetic manipulation of specific genes like mtnP.

What strategies can improve PEGylation of recombinant X. fastidiosa mtnP for increased stability in research applications?

PEGylation can significantly enhance enzyme stability and half-life for research applications. For X. fastidiosa mtnP, a rational surface engineering approach is recommended:

• Rational Surface Engineering Strategy:

  • Conduct structural analysis to identify surface-exposed amino acids suitable for PEGylation

  • Prioritize lysine residues distant from the active site

  • Identify arginine residues that could be substituted with lysine for additional PEGylation sites

  • Identify lysine residues near the active site that should be substituted with arginine to prevent activity loss

• Site-Directed Mutagenesis:

  • Design primers for site-directed mutagenesis to create the following mutants:

    • Arg→Lys substitutions at selected surface positions

    • Lys→Arg substitutions near the catalytic site

  • Confirm mutations by DNA sequencing

• PEGylation Chemistry Selection:

  • Random N-hydroxysuccinimide (NHS) ester chemistry targets primary amines (lysines)

  • Maleimide chemistry targets cysteines (if present or engineered)

  • Aldehyde chemistry targets N-terminal amines

• PEGylation Optimization:

  • Test various PEG molecule sizes (5 kDa, 10 kDa, 20 kDa, 40 kDa)

  • Optimize PEG:protein molar ratios (typically 10:1 to 100:1)

  • Adjust reaction conditions (pH 7.4-8.5, temperature, time)

  • Use size exclusion chromatography to purify PEGylated protein

• Activity Analysis:

  • Compare catalytic activity before and after PEGylation

  • Assess thermal stability of PEGylated vs. non-PEGylated enzyme

  • Evaluate pH stability profiles

  • Measure half-life in relevant buffer conditions

This approach has been shown to produce more efficient, homogeneous, and reproducible PEGylation without negatively affecting catalytic activity . In similar studies, properly engineered enzymes maintained equal levels of catalytic activity before and after PEGylation, while wild-type enzymes typically showed reduced activity following random PEGylation .

How can recombinant X. fastidiosa mtnP be used to study bacterial-plant interactions?

Recombinant X. fastidiosa mtnP can serve as a valuable tool for investigating bacterial-plant interactions through several methodological approaches:

• Gene Knockout and Complementation Studies:

  • Create mtnP deletion mutants in X. fastidiosa

  • Complement with recombinant mtnP (wild-type or catalytically inactive variants)

  • Assess changes in virulence, colonization patterns, and biofilm formation in plant hosts

  • Evaluate plant defense responses against different mutants

• Metabolic Profiling:

  • Use recombinant mtnP as a tool to analyze S-methyl-5'-thioinosine levels in plant tissues

  • Compare methionine cycle metabolite profiles between infected and uninfected plants

  • Monitor real-time changes in metabolites using microfluidic systems coupled with mass spectrometry

  • Establish correlations between metabolite levels and disease progression

• Plant-Bacterial Communication:

  • Investigate if mtnP activity affects quorum sensing molecule production

  • Determine if methionine cycle metabolites serve as signals in plant-bacterial interactions

  • Examine if plant defense compounds affect mtnP activity and bacterial metabolism

• Evolutionary Adaptation Studies:

  • Compare mtnP sequences and activities across X. fastidiosa strains with different host ranges

  • Investigate if horizontal gene transfer has shaped mtnP evolution in plant pathogens

  • Determine if recombination events in mtnP correlate with host specificity changes

• Biofilm Formation Analysis:

  • Assess the role of mtnP in biofilm formation within plant xylem vessels

  • Use fluorescently tagged recombinant mtnP to visualize localization within biofilms

  • Determine if metabolic changes due to mtnP activity influence attachment to plant surfaces

X. fastidiosa has a broad host range as a species, but individual sequence types (STs) typically cause severe disease only in a limited number of plant species . Recombination between strains may result in pathogenicity on novel hosts . Studying mtnP's role in metabolism could provide insights into these host-specificity mechanisms.

What analytical techniques are most effective for measuring the activity of recombinant X. fastidiosa mtnP?

For accurate and reproducible measurement of recombinant X. fastidiosa mtnP activity, researchers should consider these analytical approaches:

• Spectrophotometric Assays:

  • Continuous assay monitoring hypoxanthine formation at 249 nm

  • Coupled enzyme assays using xanthine oxidase to convert hypoxanthine to uric acid (monitored at 293 nm)

  • Optimization parameters:

    • Buffer composition (typically phosphate buffer, pH 7.0-7.5)

    • Temperature (25-37°C)

    • Substrate concentration range (0.01-2 mM)

    • Enzyme concentration (0.1-10 μg/ml)

• High-Performance Liquid Chromatography (HPLC):

  • Reverse-phase HPLC with C18 column

  • UV detection at 254 nm for nucleosides and bases

  • Gradient elution using methanol/water or acetonitrile/water

  • Quantification using calibration curves with pure standards

• Liquid Chromatography-Mass Spectrometry (LC-MS):

  • More sensitive detection of reaction products

  • Ability to identify and quantify multiple metabolites simultaneously

  • Multiple reaction monitoring (MRM) for specific quantification

  • Can be coupled with microfluidic systems for real-time analysis

• Radiometric Assays:

  • Use of 14C or 3H-labeled substrates

  • Separation of products by thin-layer chromatography

  • Quantification by scintillation counting

  • Provides high sensitivity for kinetic measurements

• Microfluidic Real-Time Analysis:

  • Continuous monitoring of enzyme activity in flow systems

  • Direct coupling with mass spectrometry for immediate product detection

  • Enables observation of activity changes under varying conditions

  • Allows for cellular exometabolome analysis in complex systems

When measuring enzyme kinetics, it's important to establish linear ranges for both time and enzyme concentration. A comparison of kinetic parameters (Km, kcat, kcat/Km) with orthologs provides valuable insights into substrate specificity and catalytic efficiency. For example, S-methyl-5'-thioinosine phosphorylase in P. aeruginosa has been well-characterized in its role in 5'-methylthioadenosine catabolism , providing a useful benchmark for X. fastidiosa mtnP characterization.

How does recombinant expression of X. fastidiosa mtnP compare across different expression systems?

A comparative analysis of expression systems for X. fastidiosa mtnP reveals significant differences in yield, solubility, and activity:

*Note: Values extrapolated from similar enzyme expression studies as specific data for X. fastidiosa mtnP across all these systems is not directly available in the search results.

When selecting an expression system, several factors should be considered:

• N-terminal Sequence Analysis:

  • Wild-type proteins with unfavorable N-terminal sequences often express poorly in E. coli

  • Truncation constructs should be designed based on structural analysis and sequence alignment

  • Translation initiation rate (TIR) analysis can predict construct expression levels

• Codon Optimization Strategy:

  • Codon optimization for E. coli expression often improves yield significantly

  • Different optimization algorithms produce varying results

  • Balance between high-expression codons and maintaining mRNA secondary structure

• Fusion Tag Selection:

  • His6-tag position (N- or C-terminal) affects expression and purification efficiency

  • MBP or SUMO tags can increase solubility but may affect activity

  • TEV or other protease cleavage sites should be included if tag removal is necessary

• Expression Conditions:

  • IPTG concentration optimization (typically 0.1-1.0 mM)

  • Temperature range testing (16-37°C)

  • Media composition (rich vs. minimal)

  • Induction timing and duration

N-terminal truncation strategies have been particularly effective for improving recombinant expression of difficult proteins in E. coli, with studies showing up to 28-fold improvements in yield . These improvements are often correlated with both protein sequence features (such as removal of problematic residues like consecutive prolines) and mRNA features that enhance translation initiation .

What are common challenges in purifying recombinant X. fastidiosa mtnP and how can they be addressed?

Researchers often encounter several challenges when purifying recombinant X. fastidiosa mtnP. Here are methodological solutions to these common issues:

• Low Solubility and Inclusion Body Formation:

  • Optimize expression temperature (reduce to 16-25°C)

  • Consider N-terminal truncation strategies to improve solubility

  • Add solubility-enhancing agents to lysis buffer (0.1-1% Triton X-100, 5-10% glycerol)

  • Test different host strains (e.g., Arctic Express, Rosetta)

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Low Binding Affinity to Purification Resin:

  • For His-tagged constructs, optimize imidazole concentration in binding buffer (10-40 mM)

  • Consider tag position (N- vs C-terminal) based on protein structure

  • Adjust pH and salt concentration of binding buffer

  • Test alternative affinity tags (Strep-tag II, FLAG-tag)

• Co-purification of Contaminants:

  • Implement a two-step purification strategy:

    1. Affinity chromatography (IMAC)

    2. Size exclusion or ion exchange chromatography

  • Include more stringent washing steps with higher imidazole (50-70 mM)

  • Add DNase I (10 μg/ml) and RNase A (5 μg/ml) to lysis buffer

  • Consider on-column refolding for difficult proteins

• Protein Instability During Purification:

  • Add protease inhibitors to all buffers (PMSF, EDTA, or commercial cocktails)

  • Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

  • Optimize buffer composition (50-100 mM phosphate or Tris, pH 7.0-8.0)

  • Maintain low temperature throughout purification (4°C)

  • Add stabilizing agents (5-10% glycerol, 100-200 mM NaCl)

• Loss of Activity After Purification:

  • Test activity immediately after each purification step

  • Include cofactors or metal ions if required (typically phosphate for phosphorylases)

  • Avoid freeze-thaw cycles by aliquoting purified protein

  • Consider storage conditions (buffer composition, temperature)

  • Add stabilizing agents like BSA (0.1 mg/ml) to storage buffer

Using N-terminal truncation strategies based on structural and phylogenetic analysis has been particularly effective for improving both expression and subsequent purification of recombinant proteins . These approaches can increase yield from 3-5 mg/L to much higher levels, while simultaneously improving protein solubility and reducing the formation of inclusion bodies .

How can potential contamination with E. coli phosphorylases be detected and eliminated when working with recombinant X. fastidiosa mtnP?

Contamination with endogenous E. coli phosphorylases is a significant concern when purifying recombinant X. fastidiosa mtnP. Here is a methodological approach to detect and eliminate such contamination:

• Contamination Detection Methods:

  • Mass Spectrometry Analysis:

    • Tryptic digest followed by LC-MS/MS

    • Database search against both X. fastidiosa and E. coli proteins

    • Quantification of contaminating proteins based on peptide intensity

  • Western Blot Analysis:

    • Use antibodies specific to common E. coli phosphorylases

    • Compare against purified recombinant X. fastidiosa mtnP

    • Include appropriate controls (E. coli lysate, purified E. coli phosphorylases)

  • Activity Assays with Differential Substrates:

    • Test activity against substrates specific to X. fastidiosa mtnP

    • Test activity against substrates specific to E. coli phosphorylases

    • Compare activity ratios with those of pure enzymes

• Contamination Prevention Strategies:

  • Expression System Selection:

    • Use E. coli strains with deletions in relevant phosphorylase genes

    • Consider alternative expression hosts if E. coli contamination is persistent

  • Purification Optimization:

    • Implement multi-step purification protocols:

      1. Affinity chromatography (IMAC for His-tagged proteins)

      2. Ion exchange chromatography

      3. Size exclusion chromatography

    • Optimize salt gradients for ion exchange to separate similar proteins

    • Use more stringent washing conditions during affinity purification

  • Tag Design Considerations:

    • Use dual affinity tags (His-tag plus Strep-tag II or FLAG-tag)

    • Include specific protease cleavage sites between tags and protein

    • Consider tag position based on structural differences with E. coli enzymes

• Contamination Elimination Techniques:

  • Heat Treatment:

    • If X. fastidiosa mtnP has higher thermal stability, brief heat treatment (50-60°C for 10 minutes) may selectively denature E. coli proteins

  • Substrate-Specific Elution:

    • Use competitive elution with substrates or substrate analogs specific to X. fastidiosa mtnP

  • Negative Selection:

    • Pre-adsorb lysate with antibodies against E. coli phosphorylases

    • Use immunoprecipitation to remove E. coli contaminants

• Validation of Purity:

  • Activity Ratios:

    • Calculate and compare specific activities with different substrates

    • Plot ratios to identify presence of contaminating activities

  • SDS-PAGE with Overloading:

    • Run heavily overloaded gels to detect minor contaminants

    • Use silver staining for maximum sensitivity

  • Analytical SEC-MALS:

    • Size exclusion chromatography with multi-angle light scattering

    • Confirms protein homogeneity and molecular weight

What genomic approaches could reveal the evolution of mtnP in Xylella fastidiosa strains with different host specificities?

To investigate the evolution of mtnP in X. fastidiosa strains with different host specificities, researchers could implement these genomic approaches:

• Comparative Genomic Analysis:

  • Sequence mtnP genes from multiple X. fastidiosa strains representing:

    • Different subspecies (fastidiosa, multiplex, pauca)

    • Different sequence types (STs)

    • Different host plant specificities

  • Identify single nucleotide polymorphisms (SNPs) and insertion/deletion events

  • Calculate dN/dS ratios to detect selection pressure on mtnP

• Phylogenetic Analysis:

  • Construct phylogenetic trees based on:

    • mtnP gene sequences

    • Whole-genome sequences

    • Housekeeping genes

  • Compare mtnP phylogeny with species phylogeny to detect horizontal gene transfer events

  • Calculate evolutionary distances and divergence times

• Recombination Analysis:

  • Apply recombination detection algorithms to mtnP sequences

  • Identify potential recombination breakpoints

  • Similar to how type I R-M systems in X. fastidiosa undergo recombination with exchange of target recognition domains

  • Determine if recombination events correlate with host range shifts

• Population Genetics Approach:

  • Calculate nucleotide diversity (π) and Tajima's D for mtnP across populations

  • Perform fixation index (FST) analysis to determine genetic differentiation

  • Identify structural variants in mtnP and surrounding genomic regions

  • Apply genome-wide association studies (GWAS) to correlate mtnP variants with host specificity

• Functional Genomics Integration:

  • Correlate mtnP sequence variations with:

    • Expression levels in different hosts (RNA-seq data)

    • Protein abundance (proteomics data)

    • Metabolite profiles (metabolomics data)

  • Create a systems biology model incorporating mtnP function in different strains

This approach would leverage the understanding that X. fastidiosa demonstrates significant strain variability in virulence on specific host plant species . The number of host plant species upon which a given sequence type will cause severe disease appears to be limited, and recombination between strains may result in pathogenicity on novel hosts . Analysis of 129 X. fastidiosa genome assemblies has already revealed significant genetic diversity and evidence of recombination in other systems , suggesting similar patterns may exist for metabolic genes like mtnP.

How might structural studies of X. fastidiosa mtnP inform the design of specific inhibitors as potential antimicrobials?

Structural studies of X. fastidiosa mtnP offer a foundation for rational inhibitor design, potentially leading to novel antimicrobials. A comprehensive approach would include:

• Structural Determination Methods:

  • X-ray Crystallography:

    • Express and purify recombinant X. fastidiosa mtnP in high yield

    • Screen crystallization conditions (pH, temperature, precipitants)

    • Solve structures at high resolution (<2.0 Å)

    • Co-crystallize with:

      • Natural substrates (S-methyl-5'-thioinosine)

      • Products (hypoxanthine, S-methyl-5-thio-alpha-D-ribose 1-phosphate)

      • Substrate analogs or transition state mimics

  • Cryo-Electron Microscopy:

    • Alternative approach if crystallization proves difficult

    • Capture different conformational states

    • Visualize enzyme dynamics during catalysis

  • NMR Spectroscopy:

    • For analyzing protein-ligand interactions in solution

    • Isotope labeling of recombinant protein (15N, 13C)

    • Chemical shift perturbation assays with ligands

• Computational Structure Analysis:

  • Identify Catalytic Residues:

    • Active site mapping through structure-function analysis

    • Comparison with orthologous enzymes (e.g., from P. aeruginosa)

    • Molecular dynamics simulations to identify conformational changes

  • Binding Pocket Characterization:

    • Map substrate binding sites and interactions

    • Identify specificity-determining residues

    • Calculate electrostatic potential maps

    • Perform water mapping to identify displaceable water molecules

  • Virtual Screening:

    • In silico docking of chemical libraries

    • Fragment-based screening approaches

    • Pharmacophore model development

    • Machine learning for hit prediction

• Structure-Based Inhibitor Design:

  • Transition State Analog Design:

    • Based on the enzyme's catalytic mechanism

    • Incorporation of phosphonate groups to mimic phosphate

    • Addition of leaving group mimics

  • Fragment Growing/Linking Approach:

    • Identify small molecule fragments that bind different sub-pockets

    • Link or grow fragments to create high-affinity inhibitors

  • Structure-Activity Relationship Studies:

    • Systematic chemical modifications of initial hits

    • Correlation with inhibitory potency

    • Optimization of pharmacokinetic properties

• Selectivity Analysis:

  • Structural Comparison with Human Homologs:

    • Identify structural differences for selectivity

    • Design inhibitors that exploit bacterial-specific features

    • Similar to approaches used for human thymidine phosphorylase (HsTP)

  • Cross-Species Reactivity Testing:

    • Test inhibitors against mtnP from:

      • Different X. fastidiosa strains

      • Other plant pathogens

      • Human gut microbiome bacteria

    • Balance broad-spectrum activity with specificity

This approach would leverage understanding of enzyme structure-function relationships similar to those explored for human thymidine phosphorylase (HsTP) , but with a focus on bacterial-specific features that could be exploited for antimicrobial development.