Recombinant Xylella fastidiosa Imidazole glycerol phosphate synthase subunit HisH (hisH)

What is the functional role of imidazole glycerol phosphate synthase in Xylella fastidiosa?

Imidazole glycerol phosphate synthase plays a crucial role in linking histidine and de novo purine biosynthesis pathways in Xylella fastidiosa, as it does in other bacteria. The enzyme functions as a bienzyme complex comprising the glutaminase subunit HisH and the synthase subunit HisF. In this bifunctional system, nascent ammonia produced by the HisH subunit reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide . This metabolic function is essential for bacterial survival, making it a potential target for antimicrobial development in this plant pathogen.

How does the HisH subunit from X. fastidiosa compare to HisH from other bacterial species?

While the search results don't provide direct comparative data for X. fastidiosa HisH specifically, studies of imidazole glycerol phosphate synthase in other bacteria such as Thermotoga maritima reveal conservation patterns likely applicable to X. fastidiosa. In T. maritima, isolated tHisH showed no detectable glutaminase activity on its own but was stimulated by complex formation with tHisF when either the product imidazole glycerol phosphate or a substrate analogue was bound . Based on the general conservation of this enzyme across bacterial species, X. fastidiosa HisH likely has similar functional dependencies. Researchers should note that specific structural variations may exist due to X. fastidiosa's genomic diversity and extensive evidence of recombination between subspecies .

How might intersubspecific recombination in X. fastidiosa affect the structure and function of the HisH protein?

Intersubspecific homologous recombination (IHR) has been extensively documented in X. fastidiosa populations, with significant evidence of genetic exchange between subspecies that were previously geographically isolated . This genetic recombination could potentially impact the HisH gene in several ways:

• Altered amino acid sequences resulting in modified protein-protein interactions with HisF

• Changes in enzyme kinetics or substrate specificity

• Variations in structural stability under different environmental conditions

Research has demonstrated that IHR in X. fastidiosa can be detected across multiple loci, with some sequence types showing recombination at several genetic positions . For HisH specifically, researchers would need to analyze sequence variations across different X. fastidiosa subspecies (fastidiosa, sandyi, multiplex, and pauca) to determine if this gene has been subject to recombination events. Comparative analysis of HisH sequences from different strains could reveal chimeric structures combining genetic elements from multiple subspecies, potentially creating novel functional properties that might influence host adaptation or virulence .

What are the critical catalytic residues in X. fastidiosa HisH and how can they be experimentally identified?

Based on studies of imidazole glycerol phosphate synthase in related bacteria, identifying critical catalytic residues in X. fastidiosa HisH would likely involve site-directed mutagenesis approaches similar to those used for the Thermotoga maritima enzyme. In T. maritima, eight conserved amino acids at the putative active site of tHisF were exchanged through site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics .

For X. fastidiosa HisH, a comprehensive approach would include:

• Sequence alignment with well-characterized HisH proteins to identify conserved residues

• Systematic site-directed mutagenesis of these conserved residues

• Expression and purification of mutant proteins

• Kinetic characterization to determine effects on:

  • Glutaminase activity

  • Ammonia transfer to HisF

  • Complex formation with HisF

Particular attention should be paid to residues involved in the glutaminase active site and those at the HisH-HisF interface, as these would be critical for function. For instance, in T. maritima, aspartate 11 was found to be essential for synthase activity both in vitro and in vivo, while aspartate 130 could only be partially replaced by glutamate .

How does the interaction between HisH and HisF subunits affect catalytic activity in X. fastidiosa, and what techniques can best characterize this interaction?

The interaction between HisH and HisF subunits is crucial for the function of imidazole glycerol phosphate synthase. In T. maritima, isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF when bound to either the product imidazole glycerol phosphate or a substrate analogue . This suggests a communication mechanism between the two active sites.

For X. fastidiosa, several techniques could characterize this interaction effectively:

Research should focus on how substrate binding to HisF influences HisH activity, as this allosteric communication is likely central to the synchronized function of the bienzyme complex in X. fastidiosa .

What protein purification strategy is most effective for obtaining active recombinant X. fastidiosa HisH?

For purification of recombinant X. fastidiosa HisH, a multi-step chromatographic approach would likely be most effective. Based on strategies used for similar bacterial proteins, the following protocol could be implemented:

• Affinity Chromatography:

  • Express HisH with an N- or C-terminal His-tag in E. coli

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Purify using Ni-NTA resin with imidazole gradient elution (20-250 mM)

• Ion Exchange Chromatography:

  • After affinity purification, dialyze against low-salt buffer

  • Apply to anion exchange column (Q Sepharose)

  • Elute with NaCl gradient (0-500 mM)

• Size Exclusion Chromatography:

  • Final polishing step using Superdex 75 or 200

  • Buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT

It's crucial to maintain protein stability throughout purification by including glycerol (10%) and possibly glutamine or glutamine analogues that may stabilize the protein structure. Additionally, researchers should monitor activity at each purification step, as HisH activity is dependent on interaction with HisF . Co-expression and co-purification of HisH with HisF may yield better results than purifying HisH alone, particularly for functional studies.

How can one establish a reliable enzymatic assay for X. fastidiosa HisH activity?

Establishing a reliable enzymatic assay for X. fastidiosa HisH activity presents challenges due to its functional dependence on the HisF subunit. Based on characterization of similar enzymes, a comprehensive assay system would include:

• Glutaminase Activity Assay:

  • Measure glutamine hydrolysis to glutamate and ammonia

  • Use either glutamate dehydrogenase coupled assay (monitoring NADH oxidation spectrophotometrically) or a colorimetric assay for ammonia production

  • Include purified HisF and appropriate substrate analogs to stimulate activity

• Coupled HisH-HisF Assay:

  • Monitor complete reaction from glutamine to imidazole glycerol phosphate

  • Requires synthesis or commercial source of N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR)

  • Detection of 5-aminoimidazole-4-carboxamide ribotide (AICAR) formation by HPLC or spectroscopic methods

• Control Experiments:

  • HisH alone (expected minimal activity)

  • HisH with HisF but without substrates

  • HisH with HisF and PRFAR but without glutamine

  • Use of glutamine analogs and inhibitors to confirm specificity

The assay conditions should be optimized for temperature, pH, and ionic strength, considering X. fastidiosa's natural environment. Researchers should note that, as observed with T. maritima, isolated HisH may show no detectable glutaminase activity but could be stimulated by complex formation with HisF bound to either the product or a substrate analogue .

What approaches can be used to study the impact of X. fastidiosa subspecies-specific variations in HisH on enzyme function?

To study how subspecies-specific variations in X. fastidiosa HisH affect enzyme function, researchers can employ several complementary approaches:

• Comparative Sequence Analysis:

  • Align HisH sequences from multiple X. fastidiosa subspecies (fastidiosa, sandyi, multiplex, and pauca)

  • Identify subspecies-specific polymorphisms

  • Use evolutionary analysis to distinguish between neutral variations and potentially functional mutations

• Recombinant Expression of Variant HisH Proteins:

  • Clone and express HisH from different X. fastidiosa subspecies

  • Create chimeric proteins based on observed recombination patterns in natural isolates

  • Express and purify proteins under identical conditions for direct comparison

• Functional Characterization:

  • Compare kinetic parameters (kcat, KM) of variant HisH proteins

  • Analyze thermal stability and pH optima differences

  • Assess protein-protein interaction strength with cognate HisF partners

• Structural Analysis:

  • Determine structures of variant HisH proteins by X-ray crystallography or cryo-EM

  • Map subspecies-specific variations onto structural models

  • Identify if variations cluster in functional regions (active site, HisF interface)

• In vivo Complementation Assays:

  • Test ability of variant HisH proteins to complement histidine auxotrophy in appropriate bacterial strains

  • Evaluate growth rates and fitness under various conditions

This multi-faceted approach would provide insights into how genetic recombination events between X. fastidiosa subspecies might influence HisH function and potentially contribute to adaptive processes in this plant pathogen.

What are common challenges in achieving soluble expression of recombinant X. fastidiosa HisH, and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant X. fastidiosa HisH, similar to issues documented with other bacterial enzymes:

• Inclusion Body Formation:

  • Challenge: Overexpression often leads to protein aggregation and inclusion body formation

  • Solution: Lower induction temperature (16-20°C), reduce IPTG concentration (0.1-0.2 mM), use slower-promoting media, or employ specialized E. coli strains like Arctic Express or Rosetta

• Improper Folding:

  • Challenge: Expression host may lack proper folding machinery for X. fastidiosa proteins

  • Solution: Co-express with chaperones (GroEL/GroES, DnaK/DnaJ), consider alternative expression hosts like M. smegmatis which has shown success with similar enzymes

• Limited Solubility:

  • Challenge: Even when folded correctly, protein may have limited solubility

  • Solution: Optimize buffer conditions (pH, ionic strength), add stabilizing agents (glycerol 5-10%, glutamine as natural substrate), consider fusion tags beyond His-tag (MBP, SUMO)

• Low Activity:

  • Challenge: Protein appears soluble but shows limited activity

  • Solution: Co-express with partner protein HisF, as isolated HisH typically shows no detectable glutaminase activity but is stimulated by complex formation with HisF

• Protein Degradation:

  • Challenge: Rapid degradation during expression or purification

  • Solution: Include protease inhibitors, optimize purification speed, reduce temperature during handling

A systematic approach testing multiple expression conditions is recommended, as the experience with myo-inositol-1-phosphate synthase from M. tuberculosis demonstrates that host selection can be critical—E. coli expression yielded inactive protein despite NAD+ binding, while M. smegmatis expression produced functionally active enzyme .

How can researchers address inconsistent results when comparing HisH proteins from different X. fastidiosa subspecies?

When comparing HisH proteins from different X. fastidiosa subspecies, researchers may encounter inconsistent results due to several factors:

• Genetic Recombination Effects:

  • Challenge: Natural X. fastidiosa populations show extensive evidence of intersubspecific homologous recombination

  • Solution: Thoroughly sequence and verify all cloned genes; construct phylogenetic trees to ensure proper classification of source material

• Experimental Standardization:

  • Challenge: Slight variations in purification, storage, or assay conditions can disproportionately affect different protein variants

  • Solution: Process all variants in parallel under identical conditions; include internal controls; perform technical replicates with multiple protein preparations

• Partner Protein Compatibility:

  • Challenge: HisH activity depends on interaction with HisF, which may also vary between subspecies

  • Solution: Test each HisH variant with both its cognate HisF and with HisF from other subspecies; characterize protein-protein interactions independently from enzymatic activity

• Data Analysis Approach:

  • Challenge: Statistical treatment of kinetic data may obscure or exaggerate differences

  • Solution: Employ multiple analytical methods; use global fitting approaches for complex kinetic models; validate with orthogonal techniques

• Experimental Table for Standardized Comparisons:

What are the most promising future research directions for recombinant X. fastidiosa HisH studies?

Based on the current state of knowledge, several promising research directions for recombinant X. fastidiosa HisH warrant investigation:

• Structural Biology Approaches:

  • Determination of high-resolution structures of X. fastidiosa HisH-HisF complexes from different subspecies

  • Investigation of conformational changes during catalysis using techniques like HDX-MS or FRET

  • Computational modeling of the ammonia channel between HisH and HisF active sites

• Evolutionary and Ecological Studies:

  • Analysis of HisH sequence variation across X. fastidiosa strains from different plant hosts

  • Investigation of whether recombination events in HisH contribute to host adaptation

  • Comparison with HisH from other plant pathogens to identify convergent evolutionary patterns

• Biotechnological Applications:

  • Development of HisH-based biosensors for metabolic intermediates

  • Exploration of HisH as a potential target for antimicrobial compounds specific to X. fastidiosa

  • Engineering of HisH variants with altered catalytic properties for metabolic engineering applications

• Systems Biology Integration:

  • Multi-omics approaches to understand HisH regulation in the context of X. fastidiosa metabolism

  • Quantification of metabolic flux through the histidine pathway under different conditions

  • Network analysis of interactions between histidine biosynthesis and other metabolic pathways

These research directions would not only advance fundamental understanding of X. fastidiosa biology but could also contribute to developing strategies for managing diseases caused by this economically important plant pathogen .