Recombinant Pseudomonas syringae pv. tomato Anthranilate phosphoribosyltransferase (trpD)

What is the function of TrpD in Pseudomonas syringae pv. tomato?

Anthranilate phosphoribosyltransferase (TrpD) in P. syringae pv. tomato catalyzes the second step in the tryptophan biosynthesis pathway. Specifically, it transfers the phosphoribosyl group from 5-phosphoribosyl-1-pyrophosphate (PRPP) to anthranilate, yielding N-(5'-phosphoribosyl)-anthranilate (PRA) . This reaction is essential for bacterial survival as it forms part of the critical metabolic pathway converting chorismate to tryptophan.

Recent research has revealed that TrpD also plays a secondary role in thiamine (vitamin B1) biosynthesis. Studies have demonstrated that TrpD can generate phosphoribosylamine (PRA) from enamines and PRPP, an intermediate required for thiamine synthesis . This dual functionality makes TrpD particularly significant for metabolic integration studies.

The catalytic mechanism appears to follow a dissociative pathway similar to other phosphoribosyltransferases . The enzyme's significance in bacterial metabolism is highlighted by its conservation across Pseudomonas species and its potential as an antimicrobial target due to its essentiality for bacterial growth in environments where tryptophan is limited.

How is the trpD gene organized in the P. syringae genome?

In Pseudomonas syringae pv. tomato DC3000, the trpD gene is identified with the gene ID 1182204 . While the search results don't explicitly detail the entire genomic organization in P. syringae pv. tomato, insights from related Pseudomonas species provide valuable information about likely arrangements.

Studies on P. putida, a related species, have shown that the trpD gene exists as part of the trpGDC operon . This organization, with tryptophan biosynthesis genes distributed in multiple clusters rather than a single operon, is characteristic of Pseudomonas species. The trpGDC operon contains genes encoding:

• trpG: Anthranilate synthase, component II

• trpD: Anthranilate phosphoribosyltransferase

• trpC: Indole-3-glycerol phosphate synthase

The physical organization of tryptophan biosynthesis genes in Pseudomonas typically consists of:

• Two three-gene clusters (trpGDC and trpAB/trpI)

• Two monocistronic units (trpE and trpF)

This organization differs from other bacteria such as Acinetobacter calcoaceticus and Burkholderia acidovorans, where tryptophan biosynthesis genes are arranged differently. In Pseudomonas species, the trpE gene is typically separated from the trpGDC operon by one or two genes of unknown function .

What experimental methods are optimal for expressing and purifying recombinant P. syringae TrpD?

Although the search results don't provide a specific protocol for P. syringae pv. tomato TrpD expression, general methodological approaches for recombinant expression and purification of bacterial enzymes can be applied with optimization for this specific protein.

Expression system optimization:

• Construct design:

  • Clone the trpD gene (ID: 1182204) from P. syringae pv. tomato DC3000 genomic DNA

  • Design primers with appropriate restriction sites for directional cloning

  • Insert into an expression vector with an inducible promoter (typically T7) and affinity tag (His₆ or GST)

• Expression conditions optimization:

  • Compare E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Test induction parameters (IPTG concentration: 0.1-1.0 mM)

  • Evaluate temperature conditions (37°C for 3-4 hours vs. 16-18°C overnight)

  • Consider co-expression with chaperones if folding is problematic

Purification strategy:

• Primary purification:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Buffer optimization to include Mg²⁺ (cofactor for enzymatic activity)

  • Consider including low concentrations of substrate analogues for stability

• Secondary purification:

  • Size exclusion chromatography to isolate properly folded dimeric TrpD

  • Ion exchange chromatography for removing contaminants

• Quality assessment:

  • SDS-PAGE for purity evaluation

  • Activity assays measuring conversion of anthranilate and PRPP to PRA

  • Thermal shift assays for protein stability assessment

For studying structure-function relationships, crystallization trials could be set up with purified TrpD in the presence of substrates or inhibitors, as has been done with related anthranilate phosphoribosyltransferases .

How can TrpD activity be measured in laboratory conditions?

Several complementary approaches can be employed to measure TrpD activity, depending on the research objectives and available equipment:

Direct enzymatic assays:

• Spectrophotometric/fluorometric methods:

  • Monitoring anthranilate consumption (excitation: 310 nm, emission: 400 nm)

  • Following decrease in PRPP concentration using coupled assays

  • Detection of pyrophosphate release using enzyme-coupled assays with pyrophosphatase and detection reagents

• Chromatographic techniques:

  • HPLC separation and quantification of anthranilate, PRPP, and PRA

  • LC-MS for more sensitive detection and confirmation of product identity

Genetic complementation approaches:

• In vivo functional assessment:

  • Transform trpD-deficient bacterial strains with recombinant trpD

  • Measure growth restoration in minimal media lacking tryptophan

  • Quantify tryptophan production using bioassays

A standard enzyme assay protocol might include:

• Reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂

• Substrate concentrations: 0.1-1.0 mM anthranilate, 1-5 mM PRPP

• Incubation at 25-30°C for 5-30 minutes

• Reaction termination with acid or heat

• Product detection as described above

For kinetic parameter determination, varying substrate concentrations would be used while keeping other parameters constant. When studying the alternative TrpD activity in thiamine synthesis, isotopic labeling can be employed to trace the conversion of enamines to phosphoribosylamine .

What structural features determine TrpD substrate binding and catalysis?

Anthranilate phosphoribosyltransferase (TrpD) has a distinctive structure with key features that determine its substrate binding and catalytic mechanism:

• Homodimeric enzyme with the active site located in the hinge region between two domains

• Each monomer contains distinct domains responsible for binding different substrates

Substrate binding sites:

• PRPP binding:

  • Binds to the C-terminal domain

  • Coordinates to Mg²⁺, which is essential for activity

  • Binding site is completed by two flexible loops

  • Conserved residues interact with the phosphate and ribose moieties

• Anthranilate binding:

  • Features multiple binding sites along an "anthranilate channel"

  • This multi-modal binding explains substrate inhibition at high anthranilate concentrations

  • Binding involves both hydrophobic interactions and specific polar contacts

Catalytic mechanism:

• Follows a dissociative mechanism similar to other phosphoribosyltransferases

• Involves formation of a ribosyl carbonium ion intermediate

• Nucleophilic attack by anthranilate on this intermediate forms the PRA product

The multi-binding mode for anthranilate has implications for inhibitor design, as the best inhibitors exploit these multiple binding sites . For the secondary activity in thiamine synthesis, TrpD can accommodate 4- and 5-carbon enamines as substrates instead of anthranilate, with variants affecting substrate binding residues impacting this alternative activity while retaining the primary function .

Crystal structures of anthranilate phosphoribosyltransferase with substrates, substrate analogues, and inhibitors have provided valuable insights into these structural features and the catalytic mechanism .

What is the evidence for TrpD's role in generating PRA for thiamine synthesis?

Recent research has uncovered a novel secondary function of TrpD in thiamine biosynthesis, distinct from its primary role in tryptophan synthesis. The evidence for this function comes from detailed biochemical and genetic studies:

Biochemical evidence:

• Substrate flexibility:

  • TrpD can utilize 4- and 5-carbon enamines as substrates instead of anthranilate

  • The reaction product is predicted to be a phosphoribosyl-enamine adduct

• Product identification:

  • Isotopic labeling studies demonstrated that the TrpD reaction product is hydrolyzed to phosphoribosylamine (PRA)

  • PRA is a known intermediate metabolite required for both purine and thiamine synthesis

Genetic evidence:

• Structure-function relationships:

  • TrpD variants with substitutions at residues involved in binding anthranilate or PRPP were proficient for tryptophan synthesis but unable to support PRA formation in vivo in Salmonella enterica

  • This suggests the canonical and alternative activities share the same active site but with different substrate specificities

• Metabolic context:

  • The alternative activity becomes significant in the absence of reactive intermediate deaminase RidA, where enamines accumulate

  • This reveals a metabolic robustness where one enzyme can compensate for metabolic imbalances

This discovery highlights how well-characterized biosynthetic enzymes can have "moonlighting" functions that contribute to metabolic network robustness. The ability of TrpD to generate PRA represents an alternative route for thiamine biosynthesis, potentially important under specific growth conditions or metabolic states.

How do mutations in trpD affect P. syringae virulence and fitness?

Impact on bacterial growth:

• Auxotrophy development:

  • Studies in related Pseudomonas species (P. putida) show that trpD mutations result in tryptophan auxotrophy

  • TrpD-deficient mutants could grow only when media was supplemented with tryptophan or indole

• Growth environment restrictions:

  • TrpD mutants would likely be severely limited in environments where tryptophan is scarce

  • This includes the plant apoplastic space during infection, which typically has limited free amino acids

Potential virulence implications:

• Infection process constraints:

  • The ability to synthesize essential amino acids is critical for successful plant colonization

  • Tryptophan auxotrophy would likely impair bacterial multiplication in planta

• Metabolic burden considerations:

  • Even if some tryptophan is available in host tissues, reliance on uptake rather than synthesis creates a competitive disadvantage

  • Energy typically used for virulence factor production might be diverted to amino acid acquisition

• Secondary effects:

  • Disruption of TrpD's alternative function in generating PRA for thiamine synthesis could further compromise bacterial fitness

  • This dual impact on both tryptophan and thiamine pathways could have synergistic negative effects

For researchers investigating trpD mutations, comprehensive approaches would include:

• Constructing defined deletion or point mutants

• Comparative growth analyses in various media with different tryptophan availability

• Plant infection assays comparing wild-type and mutant strains

• In planta competition assays between wild-type and mutant strains

• Transcriptomic analysis to identify compensatory responses to trpD mutation

How has recombination influenced the evolution of trpD in P. syringae populations?

Recombination has played a significant role in shaping P. syringae evolution, with implications for metabolic genes including trpD:

Evidence for recombination in P. syringae:

• Population genetic analyses:

  • Studies on P. syringae pv. tomato found that recombination contributed more than mutation to variation between isolates

  • Several recombination breakpoints were detected within sequenced gene fragments

• Genomic impact:

  • Recombination may play an important role in the reassortment of genes between P. syringae strains

  • This process likely contributes to metabolic adaptation and host range expansion

Implications for trpD evolution:

• Phylogenetic patterns:

  • P. syringae pv. tomato strain DC3000 clusters with isolates from several plant families and has a wider host range than typical tomato isolates

  • This suggests that recombination events might have influenced metabolic capabilities, potentially including tryptophan biosynthesis

• Functional consequences:

  • Recombination could introduce variants of trpD with altered catalytic properties

  • Such variants might affect both the primary function in tryptophan biosynthesis and the secondary role in thiamine synthesis

• Host adaptation factors:

  • The clustering of P. syringae pv. tomato DC3000 with isolates from diverse hosts suggests recombination may contribute to host range expansion

  • Metabolic gene variants acquired through recombination could facilitate adaptation to different plant nutrient environments

For metabolic genes like trpD, recombination provides a mechanism for rapid adaptation without the accumulation of potentially deleterious mutations in essential pathways. This process may be particularly important for plant pathogens like P. syringae that must adapt to diverse host environments with different nutrient availabilities.

How conserved is TrpD across different Pseudomonas species?

TrpD shows considerable conservation across Pseudomonas species, reflecting its essential role in tryptophan biosynthesis:

Sequence conservation:

• Amino acid similarity:

  • Tryptophan biosynthesis enzymes in Pseudomonas species show amino acid sequence identity in the range of 71-97%

  • This high degree of conservation suggests strong selection pressure to maintain function

• Structural conservation:

  • The catalytic core and substrate binding residues show particularly high conservation

  • This reflects the fundamental importance of the reaction catalyzed by TrpD

Genomic organization conservation:

• Gene clustering patterns:

  • The physical organization of trp genes is conserved across sequenced Pseudomonas strains

  • The trpGDC genes typically form an operon in Pseudomonas species

• Genomic context:

  • In all sequenced Pseudomonas strains, the trpE gene is separated from the trpGDC operon by one or two genes of unknown function

  • The trpF gene is consistently flanked by truA and accD genes across Pseudomonas species

This conservation table for TrpD shows representative identity percentages between selected Pseudomonas species based on information in the search results:

The high conservation of TrpD across Pseudomonas species highlights its fundamental importance in bacterial metabolism and suggests that any sequence variations might reflect fine-tuning to specific ecological niches rather than major functional differences.

What are the predicted functional interactions of TrpD with other proteins in P. syringae?

According to STRING database analysis, TrpD in P. syringae has several high-confidence predicted functional interactions with other proteins, particularly those involved in the tryptophan biosynthesis pathway:

Highest confidence interactions:

• Core tryptophan pathway enzymes:

  • TrpG (Anthranilate synthase, component II) - Score: 0.999

  • TrpC (Indole-3-glycerol phosphate synthase) - Score: 0.998

  • TrpE (Anthranilate synthase, component I) - Score: 0.997

  • TrpF (N-(5'phosphoribosyl)anthranilate isomerase) - Score: 0.997

• Additional tryptophan pathway components:

  • TrpB (Tryptophan synthase, beta subunit) - Score: 0.974

  • PabB (Para-aminobenzoate synthase, component I) - Score: 0.961

  • TrpA (Tryptophan synthase, alpha subunit) - Score: 0.959

Functional significance of interactions:

• Metabolic channeling:

  • TrpD directly utilizes anthranilate produced by TrpE/TrpG

  • The PRA product from TrpD serves as substrate for TrpF

  • These sequential enzymatic reactions may involve direct protein-protein interactions for efficient intermediate transfer

• Co-regulation:

  • High confidence scores may reflect coordinated expression

  • The organization of trpD with trpG and trpC in an operon suggests transcriptional co-regulation

• Evolutionary constraints:

  • Functional interactions impose co-evolutionary pressure

  • This explains the high conservation of these proteins across Pseudomonas species

These interaction predictions align with the genomic organization of trp genes in Pseudomonas species, where trpD is found in the trpGDC operon . The high confidence scores across multiple enzymes in the pathway suggest that TrpD functions as part of a coordinated biosynthetic network rather than in isolation.

What approaches can be used to develop inhibitors targeting P. syringae TrpD?

Targeting TrpD for inhibitor development represents a promising approach for controlling P. syringae infections in plants. Several strategic approaches can be employed:

Structure-based design strategies:

• Targeting unique binding sites:

  • Focus on the distinctive anthranilate channel with multiple binding sites

  • The best inhibitors reported to date exploit these multiple binding sites for anthranilate

• Rationale from existing structures:

  • Crystal structures of anthranilate phosphoribosyltransferase with various ligands provide templates for inhibitor design

  • Transition state analogues mimicking the dissociative reaction mechanism could be highly effective

Compound screening approaches:

• High-throughput screening:

  • Develop fluorescence-based assays monitoring anthranilate consumption

  • Screen compound libraries against purified recombinant TrpD

  • Confirm hits with secondary assays and structural studies

• Fragment-based screening:

  • Identify small molecules that bind to different sites within the enzyme

  • Link fragments to create higher-affinity inhibitors

  • Use thermal shift assays to identify stabilizing fragments

Target validation considerations:

• Essentiality confirmation:

  • Genetic studies in P. putida confirm that trpD mutants are tryptophan auxotrophs

  • Similar essentiality likely exists in P. syringae, especially during plant infection

• Specificity engineering:

  • Design inhibitors that exploit differences between plant and bacterial phosphoribosyltransferases

  • Target the unique multi-modal anthranilate binding of TrpD

• Delivery strategies for plant protection:

  • Formulate inhibitors for foliar application

  • Consider systemic delivery through plant vascular systems

  • Design pro-inhibitors activated by plant or bacterial enzymes

As TrpD is part of an essential biosynthetic pathway and shows distinct structural features, it represents a valuable target for developing new agricultural antimicrobials against P. syringae infections.

How does TrpD contribute to the host adaptation of P. syringae?

While the search results don't directly address TrpD's specific role in host adaptation, several lines of evidence suggest potential contributions:

Host range determinants:

• Metabolic flexibility:

  • P. syringae pv. tomato strain DC3000 has an unusually wide host range including tomato, Arabidopsis thaliana, and cauliflower

  • Most other tomato isolates form a distinct cluster that is pathogenic only on tomato

  • This suggests that metabolic capabilities, potentially including efficient tryptophan biosynthesis, may contribute to host range expansion

• Nutritional adaptation:

  • Different plant hosts provide varying levels of amino acids in apoplastic fluids

  • Efficient tryptophan biosynthesis would be particularly important in hosts with limited free tryptophan

  • The dual functionality of TrpD in both tryptophan and thiamine synthesis pathways may provide metabolic advantages in certain host environments

Evolutionary considerations:

• Recombination effects:

  • Recombination has contributed more than mutation to variation between P. syringae isolates

  • This could affect metabolic genes like trpD, potentially influencing adaptation to different hosts

  • The organization of trpD in an operon with other tryptophan biosynthesis genes might facilitate co-evolution of functionally related genes

• Selection pressures:

  • Different plant hosts may impose different selection pressures on tryptophan biosynthesis

  • TrpD variants could potentially be selected for optimal performance in specific host environments

The dual role of TrpD in both tryptophan and thiamine synthesis may provide metabolic flexibility that contributes to the ability of P. syringae to colonize different plant hosts. This metabolic versatility, combined with the pathogen's type III secretion system for delivering effector proteins , likely enables successful adaptation to various plant environments.

What are the kinetic parameters of P. syringae TrpD and how do they compare with other bacterial TrpDs?

While the search results don't provide specific kinetic parameters for P. syringae TrpD, we can outline the typical parameters that would be assessed and how they might compare with other bacterial TrpDs:

Key kinetic parameters for TrpD characterization:

Comparative considerations:

For researchers investigating P. syringae TrpD kinetics, enzyme assays should be performed under standardized conditions (pH 7.5-8.0, 25-30°C, 5 mM Mg²⁺) to enable direct comparison with published values for other bacterial TrpDs. Additionally, the potential for substrate inhibition should be explicitly examined by testing a wide range of anthranilate concentrations.

What methods are used to study the regulation of trpD expression in P. syringae?

While the search results don't specifically address methods for studying trpD regulation in P. syringae, several approaches can be inferred from research on related systems:

Transcriptional analysis methods:

• Quantitative techniques:

  • RT-qPCR for measuring trpD transcript levels under different conditions

  • RNA-Seq for genome-wide expression analysis including trpD

  • Northern blotting for detecting specific trpD transcripts and operon structure

• Promoter analysis approaches:

  • Reporter gene fusions (lacZ, gfp) to trpD promoter regions

  • Primer extension and 5' RACE to map transcription start sites

  • ChIP-seq to identify transcription factor binding sites

Regulatory element identification:

• Genetic approaches:

  • Deletion analysis of promoter regions to identify regulatory elements

  • Site-directed mutagenesis of potential regulatory sequences

  • Isolation of regulatory mutants with altered trpD expression

• Biochemical methods:

  • Electrophoretic mobility shift assays (EMSA) to detect protein-DNA interactions

  • DNase I footprinting to precisely map binding sites

  • In vitro transcription assays with purified components

Regulatory network analysis:

• System-level approaches:

  • Transcriptomics under various environmental conditions

  • Perturbation studies with regulatory gene knockouts

  • Network inference from large-scale expression datasets

Research in P. putida has shown that the trpD gene is part of the trpGDC operon , suggesting coordinated regulation of these genes. Additionally, the search results mention AefR as a transcriptional regulator in P. syringae pv. phaseolicola that influences type III secretion system gene induction , suggesting complex regulatory networks that might impact trpD expression during host interaction.

For comprehensive analysis of trpD regulation, researchers should examine expression under conditions relevant to the bacterium's lifecycle, including minimal vs. rich media, plant extract exposure, and various stress conditions.

How can CRISPR-Cas9 technology be applied to study TrpD function in P. syringae?

CRISPR-Cas9 technology offers powerful approaches for studying TrpD function in P. syringae through precise genetic manipulation:

Gene editing applications:

• Knockout strategies:

  • Complete deletion of trpD to create auxotrophs for complementation studies

  • Introduction of premature stop codons to generate truncated proteins

  • Targeted disruption of specific domains to dissect structure-function relationships

• Precise mutations:

  • Introduction of point mutations to alter catalytic residues

  • Modification of substrate binding sites to study specificity determinants

  • Creation of mutations to separate primary (tryptophan synthesis) and secondary (thiamine synthesis) functions

Regulatory studies:

• Promoter modifications:

  • Targeted changes to regulatory elements controlling trpD expression

  • Introduction of inducible promoters for controlled expression

  • Creation of reporter fusions at the native locus

• Operon structure analysis:

  • Disruption of operon structure to study co-regulation with trpG and trpC

  • Introduction of terminators or additional promoters within operons

Advanced applications:

• CRISPRi (CRISPR interference):

  • Reversible repression of trpD expression using catalytically inactive Cas9 (dCas9)

  • Tunable repression through modified guide RNAs

  • Temporal control of expression during different infection stages

• CRISPRa (CRISPR activation):

  • Upregulation of trpD expression to assess metabolic effects

  • Controlled overexpression to study potential feedback inhibition

• Multiplexed editing:

  • Simultaneous modification of trpD and interacting genes

  • Creation of multiple variants in parallel for comparative analysis

For implementing CRISPR-Cas9 systems in P. syringae, researchers should consider:

• Selection of appropriate Cas9 variants optimized for GC-rich Pseudomonas genomes

• Development of efficient delivery methods (electroporation, conjugation)

• Use of temperature-sensitive plasmids for transient Cas9 expression

• Implementation of appropriate selection/counter-selection strategies

• Verification of edits through sequencing and phenotypic characterization