Recombinant Bacillus subtilis Uncharacterized isochorismatase family protein yddQ (yddQ)

What is the YddQ protein in Bacillus subtilis?

YddQ is classified as a putative hydrolase in Bacillus subtilis that belongs to the isochorismatase protein family. It remains largely uncharacterized despite being conserved across multiple bacterial species. The protein is identified primarily through sequence homology and conserved structural motifs rather than experimental characterization of its function . Like approximately 25% of proteins in even well-studied model organisms like B. subtilis, YddQ lacks a definitively established function, making it part of the category of "understudied proteins" that limit our complete understanding of cellular requirements for life . Current evidence for its classification is considered level 3 (function proposed based on presence of conserved amino acid motif, structural feature or limited homology) .

Why is YddQ specifically classified within the isochorismatase family?

YddQ is classified within the isochorismatase family based on sequence analysis revealing characteristic conserved domains and catalytic residues typical of this enzyme family. Isochorismatases typically catalyze the hydrolysis of isochorismate to 2,3-dihydroxybenzoate and pyruvate, playing roles in various metabolic pathways including siderophore biosynthesis. The protein contains the conserved catalytic triad commonly found in isochorismatase family members, though its specific substrate preference remains experimentally unverified . Sequence alignment with characterized isochorismatases like YaaI (another B. subtilis isochorismatase family member) shows conservation of key structural features, supporting this classification despite limited functional characterization .

What experimental systems are available for studying YddQ?

B. subtilis serves as an excellent expression host for studying YddQ due to its GRAS (Generally Recognized as Safe) status and remarkable ability to absorb and incorporate exogenous DNA into its genome . Several expression systems can be utilized for YddQ studies, including:

• Plasmid-based expression systems with inducible promoters

• Genomic integration approaches for stable expression

• Secretion systems utilizing signal peptides for extracellular production

The choice of system depends on research objectives. For structural studies, high-yield intracellular expression is preferable, while functional studies might benefit from secreted expression to facilitate protein purification. B. subtilis offers advantages over E. coli for studying native B. subtilis proteins like YddQ as it provides the natural cellular environment and relevant post-translational modifications .

What are the predicted functional partners of YddQ?

Based on systematic protein interaction studies, YddQ has several predicted functional partners that provide clues to its potential biological role. These associations are primarily determined through computational predictions and high-throughput experimental approaches rather than targeted studies. The table below summarizes the top predicted functional partners of YddQ based on available data:

These associations suggest YddQ may function in metabolic pathways related to aromatic compound processing, potentially in secondary metabolite production or stress response mechanisms . The association with other isochorismatase family members indicates possible functional redundancy or specialization within metabolic pathways.

What bioinformatic approaches are most effective for predicting YddQ function?

Multiple bioinformatic approaches can be integrated to predict YddQ's function with increasing confidence:

• Sequence-based analysis: Beyond basic homology searching, advanced sequence analysis including detection of conserved motifs, phylogenetic profiling, and genomic context analysis can provide insights into functional conservation patterns.

• Structural prediction: AlphaFold2 or similar tools can generate structural models of YddQ, enabling identification of potential substrate binding pockets and catalytic sites. Structural comparison with characterized isochorismatases can provide mechanistic insights.

• Gene neighborhood analysis: Examining genes adjacent to yddQ can reveal functional associations, as genes involved in the same pathway are often clustered in bacterial genomes.

• Co-expression network analysis: Identifying genes with similar expression patterns across various conditions can place YddQ within specific cellular processes.

• Metabolic pathway mapping: Integrating YddQ into the metabolic network of B. subtilis based on its predicted enzymatic class can identify potential pathways where it participates .

These approaches should be used complementarily, as each has limitations when applied to understudied proteins .

How does YddQ compare to other isochorismatase family proteins?

YddQ shares key structural features with other isochorismatase family proteins while exhibiting distinctive characteristics that may indicate functional specialization. Comparative analysis with YaaI, another B. subtilis isochorismatase family protein that has received more research attention, reveals:

The similarities suggest conservation of basic enzymatic mechanism, while differences in expression patterns and potential regulatory elements may indicate different physiological roles. This comparative analysis can guide hypothesis formation for experimental validation .

What experimental approaches are most effective for determining the substrate specificity of YddQ?

Determining substrate specificity of uncharacterized enzymes like YddQ requires a multi-faceted approach:

• Targeted enzymatic assays: Based on the isochorismatase classification, screening YddQ against isochorismate and structurally related compounds (such as chorismate, prephenate, and various benzoate derivatives) using HPLC or spectrophotometric detection of reaction products.

• Metabolomic approaches: Comparing metabolite profiles between wild-type and yddQ deletion/overexpression strains using LC-MS to identify accumulated substrates or depleted products.

• Activity-based protein profiling: Using chemical probes designed to react with the catalytic residues of hydrolases to identify active site interactions.

• Crystallographic studies with substrate analogs: Co-crystallizing YddQ with substrate analogs or inhibitors to elucidate binding pocket specificity.

• Isothermal titration calorimetry (ITC): Measuring binding affinities for potential substrates to identify the most likely candidates for enzymatic activity.

These approaches should be performed iteratively, with results from each method informing the design of subsequent experiments. Additionally, comparing reaction conditions (pH, temperature, cofactor requirements) with those of characterized isochorismatases can provide insights into YddQ's specific catalytic properties .

How can protein-protein interaction studies help elucidate the function of YddQ?

Protein-protein interaction (PPI) studies are particularly valuable for elucidating the function of uncharacterized proteins like YddQ by placing them in a functional context within cellular pathways. Several methodological approaches can be employed:

• Affinity purification coupled with mass spectrometry (AP-MS): Using tagged YddQ to capture interaction partners from B. subtilis lysates, followed by MS identification. This approach can reveal both stable and transient interactions.

• Bacterial two-hybrid (B2H) screening: Systematic screening against a B. subtilis genomic library to identify binary interactions. This method is particularly useful for detecting direct interactions.

• Proximity-dependent biotin identification (BioID): Fusing YddQ to a promiscuous biotin ligase to biotinylate proteins in close proximity, revealing the spatial context of YddQ in the cell.

• Co-immunoprecipitation with targeted candidates: Testing interactions with predicted partners based on bioinformatic analysis, particularly focusing on metabolic enzymes involved in aromatic amino acid biosynthesis or related pathways.

• In situ crosslinking: Capturing transient interactions in their native cellular environment before cell disruption.

The interpretation of PPI data requires careful validation through reciprocal interactions, functional assays, and correlation with co-expression data. These approaches have been successfully applied to other understudied proteins in B. subtilis, revealing functional connections otherwise missed by sequence analysis alone .

What role might YddQ play in B. subtilis metabolic pathways?

Based on its classification as an isochorismatase family protein, YddQ likely functions within pathways related to aromatic compound metabolism. Potential metabolic roles include:

• Secondary metabolite biosynthesis: Isochorismatases often participate in the biosynthesis of siderophores, antibiotics, or other secondary metabolites derived from the shikimate pathway.

• Aromatic amino acid metabolism: YddQ may be involved in alternative pathways for processing intermediates in phenylalanine, tyrosine, or tryptophan biosynthesis or degradation.

• Stress response mechanisms: Expression pattern analysis suggests potential upregulation under specific stress conditions, indicating a role in adaptation to environmental challenges.

• Metabolic homeostasis: Like other understudied but highly expressed proteins, YddQ may function in maintaining metabolic balance under specific growth conditions.

To experimentally validate these hypotheses, metabolic flux analysis comparing wild-type and yddQ mutant strains under various conditions is recommended. Stable isotope labeling approaches can trace the specific reactions catalyzed by YddQ within these pathways. Additionally, phenotypic characterization of deletion mutants under different nutrient limitations or stress conditions may reveal conditions where YddQ function becomes essential or advantageous .

How can CRISPR-Cas9 gene editing techniques be applied to study YddQ function?

CRISPR-Cas9 technology offers powerful approaches for investigating uncharacterized proteins like YddQ through precise genetic manipulation:

• Knockout studies with phenotypic analysis: Creating clean yddQ deletion mutants without polar effects on neighboring genes, followed by comprehensive phenotyping under diverse conditions to identify growth, morphology, or stress response phenotypes.

• CRISPRi for conditional depletion: Using catalytically inactive Cas9 (dCas9) to reversibly repress yddQ expression, allowing assessment of essential functions and temporal requirements.

• Precise domain modifications: Introducing targeted mutations in catalytic residues or specific domains to assess their contribution to function while maintaining protein expression.

• Promoter replacement: Substituting the native yddQ promoter with controllable promoters to study the effects of altered expression levels.

• Tagging at native locus: Adding fluorescent or affinity tags to the endogenous yddQ gene to study localization, dynamics, and interactions without disrupting native regulation.

• Multiplexed editing: Simultaneously targeting yddQ and related isochorismatase family genes to overcome potential functional redundancy.

To maximize the utility of these approaches, CRISPR-based modifications should be combined with multi-omics profiling (transcriptomics, metabolomics) to comprehensively assess the impact of yddQ manipulation on cellular physiology. This strategy has proven effective for functional characterization of other understudied genes in B. subtilis .

What expression systems are most effective for recombinant production of YddQ in B. subtilis?

Selecting an optimal expression system for YddQ requires balancing yield, solubility, and maintenance of native structure. Based on successful approaches with similar proteins in B. subtilis, the following systems are recommended:

For initial characterization, a dual approach is recommended:

• Intracellular expression using the pHT01 system with a C-terminal His-tag for purification and biochemical characterization

• Secretory expression using pHT43 for functional assays requiring native folding

Critical parameters to optimize include:

• Induction timing (mid-log phase typically optimal)

• Induction temperature (30°C often balances yield and solubility)

• Media composition (particularly for metal-dependent activity)

For difficult expression cases, co-expression with molecular chaperones may enhance solubility, as has been demonstrated for other hydrolases in B. subtilis .

What purification strategy is most suitable for recombinant YddQ?

A multi-step purification strategy tailored to YddQ's predicted properties as an isochorismatase family protein can maximize yield, purity, and activity retention:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged YddQ, with optimization of imidazole concentration in wash and elution buffers to minimize non-specific binding common with hydrolases.

• Intermediate purification: Ion exchange chromatography based on YddQ's predicted isoelectric point (typically anion exchange at pH 8.0), which effectively separates contaminating proteins with similar affinity for IMAC.

• Polishing step: Size exclusion chromatography to remove aggregates and achieve high purity for structural studies or sensitive enzymatic assays.

Buffer optimization is critical throughout the purification process, particularly:

• Including reducing agents (1-5 mM DTT or TCEP) to maintain any catalytic cysteine residues

• Testing stability with various salt concentrations (typically 100-300 mM NaCl)

• Evaluating pH stability range (typically pH 7.0-8.0 for isochorismatases)

• Assessing the need for divalent metal ions (Mg²⁺, Mn²⁺) which may be required for activity

For activity assays, it's advisable to retain samples from each purification step to track specific activity and identify potential activity losses during purification. This approach allows optimization of conditions to maintain enzymatic function while achieving high purity .

How can structural studies contribute to understanding the catalytic mechanism of YddQ?

Structural characterization of YddQ can provide crucial insights into its catalytic mechanism and substrate specificity through multiple complementary approaches:

• X-ray crystallography: Obtaining high-resolution structures of YddQ in apo form and in complex with potential substrates or substrate analogs. Critical crystallization parameters include:

  • Protein concentration (typically 5-15 mg/ml)

  • Buffer composition (screening various pH values and salt concentrations)

  • Precipitant type and concentration

  • Additive screening (particularly including potential cofactors)

• NMR spectroscopy: For studying the dynamics of YddQ-substrate interactions, particularly if crystallization proves challenging. ¹⁵N and ¹³C labeling of recombinant YddQ would be required for detailed structural analysis.

• Cryo-electron microscopy: Particularly valuable if YddQ forms larger complexes with interaction partners, though challenging for smaller proteins like YddQ unless part of a larger assembly.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To map conformational changes upon substrate binding, providing insights into the catalytic mechanism.

• Site-directed mutagenesis of predicted catalytic residues: Based on structural information and homology to characterized isochorismatases, followed by activity assays to confirm their role in catalysis.

The structural data should be interpreted in the context of sequence conservation across the isochorismatase family and compared with structures of characterized family members. This approach allows identification of:

• Conserved catalytic residues

• Substrate binding pocket variations that may indicate different substrate preferences

• Potential allosteric sites

• Structural features that might be involved in protein-protein interactions

Combined with computational approaches like molecular docking and molecular dynamics simulations, these structural studies can generate testable hypotheses about YddQ's specific catalytic mechanism and substrate profile .

How can transcriptomic data help characterize the physiological role of YddQ?

Transcriptomic analysis can reveal the physiological contexts in which YddQ functions by identifying conditions that affect its expression and correlated gene expression patterns:

• Differential expression analysis: Comparing yddQ expression across various growth conditions (nutrient limitations, stress conditions, growth phases) to identify stimuli that regulate its expression. RNA-seq provides quantitative data on expression changes, while techniques like qRT-PCR can validate findings for specific conditions.

• Co-expression network analysis: Identifying genes whose expression patterns correlate with yddQ across multiple conditions, potentially revealing functional associations not detectable through sequence analysis alone.

• Regulon analysis: Determining if yddQ is part of known regulons by examining expression changes in response to deletion of specific transcription factors.

• Comparative transcriptomics: Analyzing expression differences between wild-type and yddQ knockout strains to identify compensatory responses that may indicate the pathways affected by YddQ function.

• Time-course transcriptomics: Following expression dynamics during transitions between conditions to place YddQ function within temporal response programs.

The resulting data can be visualized using heatmaps of co-expressed genes and pathway enrichment analysis to identify biological processes potentially linked to YddQ function. This approach has successfully revealed functional contexts for other uncharacterized proteins in B. subtilis by placing them within specific stress responses or metabolic adaptations .

What are the implications of YddQ conservation across bacterial species?

The evolutionary conservation pattern of YddQ across bacterial species provides valuable insights into its functional importance and potential specialization:

• Phylogenetic distribution analysis: Determining which bacterial lineages contain YddQ homologs can reveal whether it represents a core bacterial function or a specialized adaptation. Broad conservation suggests fundamental importance, while restricted distribution may indicate specialized functions.

• Synteny analysis: Examining the genomic context of yddQ homologs across species. Conservation of neighboring genes strongly suggests functional relationships and participation in the same pathway.

• Selection pressure analysis: Calculating dN/dS ratios to determine whether YddQ is under purifying selection (suggesting conserved function) or positive selection (suggesting adaptation to different niches).

• Domain architecture comparison: Identifying whether YddQ homologs maintain the same domain organization or acquire additional domains in specific lineages, which could indicate functional specialization.

• Correlation with ecological niches: Analyzing whether YddQ conservation correlates with specific bacterial lifestyles (soil-dwelling, pathogenic, extremophilic) to generate hypotheses about its environmental relevance.

This evolutionary perspective can guide functional studies by suggesting conditions relevant to YddQ's natural role and identifying model organisms beyond B. subtilis where complementary studies might be informative. The isochorismatase family shows interesting evolutionary patterns, with many bacteria containing multiple family members with presumed functional specialization .

What are the most promising future research directions for understanding YddQ function?

Future research on YddQ should integrate multiple approaches to overcome the challenges inherent in characterizing understudied proteins:

• Integration of multi-omics data: Combining transcriptomic, proteomic, metabolomic, and interactomic data to place YddQ within the broader cellular context, particularly focusing on stress conditions where expression may be upregulated.

• Development of high-throughput substrate screening: Establishing assays that can test YddQ activity against libraries of potential substrates, particularly focusing on metabolites in aromatic amino acid metabolism and secondary metabolite biosynthesis.

• In situ functional studies: Using approaches like proximity labeling to identify molecules that interact with YddQ in its native cellular environment, providing clues to its natural substrates.

• Systematic phenotyping: Comprehensive characterization of yddQ deletion and overexpression strains across diverse growth conditions, stresses, and nutrient sources to identify conditions where YddQ function becomes important.

• Structural biology approaches: Obtaining high-resolution structures of YddQ alone and in complex with potential substrates or inhibitors to understand its catalytic mechanism and substrate specificity.

• Synthetic biology applications: Exploring whether YddQ can be repurposed for biotechnological applications, particularly if it demonstrates activity on industrially relevant substrates.

These directions should be pursued in parallel, with regular integration of findings to refine hypotheses and guide subsequent experiments. The research community studying B. subtilis continues to develop innovative approaches for characterizing understudied proteins, which can be adapted for YddQ investigation .