Recombinant Vibrio vulnificus D-tyrosyl-tRNA (Tyr) deacylase (dtd)

What is the primary function of DTD in Vibrio vulnificus?

DTD in V. vulnificus, like in other organisms, functions primarily as an editing enzyme that hydrolyzes the ester bond formed between D-amino acids and tRNA molecules. This enzymatic activity is essential for preventing the incorporation of D-amino acids into proteins during translation, thus maintaining translational fidelity. The enzyme specifically recognizes and cleaves D-amino acids attached to tRNAs while sparing L-amino acids, which are the correct substrates for protein synthesis . This chiral discrimination is vital for V. vulnificus survival, as the incorporation of D-amino acids would lead to the production of non-functional or toxic proteins.

How does DTD's structure relate to its function in maintaining translational accuracy?

DTD from V. vulnificus likely shares the dimeric structure observed in other bacterial DTDs, with each subunit contributing to the active site. The enzyme uses an invariant "cross-subunit" Gly-cisPro dipeptide to capture the chiral center of D-aminoacyl-tRNA . This unique structural arrangement allows for:

• Recognition of the D-configuration of amino acids

• Side chain-independent substrate binding, enabling action on multiple D-amino acids

• Stereospecific exclusion of L-amino acids

The active site architecture likely contains a "SQFT" motif (similar to E. coli DTD) where threonine functions as the main nucleophile that attacks the carbonyl group of the D-amino acid linked to tRNA . The enzyme's structure enables precise positioning of the substrate for optimal hydrolysis without direct participation of protein side chains in the catalytic mechanism.

Beyond chiral proofreading, what other roles might DTD play in V. vulnificus?

Recent research has revealed that DTD functions extend beyond simple chiral proofreading. DTD has been shown to eliminate glycine mistakenly attached to tRNA^Ala^, demonstrating a role in correcting achiral amino acid mischarging . This additional function suggests DTD serves as a more general quality control enzyme in translation.

In V. vulnificus, this broader function may be particularly important due to the bacterium's need to adapt to changing environmental conditions, including varying temperatures and salinity levels in coastal waters . The dual role of DTD may contribute to V. vulnificus' ability to maintain protein synthesis fidelity under stress conditions, potentially contributing to its virulence and pathogenicity.

What are the most effective expression systems for producing recombinant V. vulnificus DTD?

Based on protocols used for DTDs from other organisms, recombinant V. vulnificus DTD can be efficiently expressed in E. coli expression systems. The recommended expression protocol includes:

• Cloning the dtd gene from V. vulnificus genomic DNA into an expression vector (e.g., pET series)

• Transforming the construct into an E. coli expression strain (BL21(DE3) or similar)

• Growing cultures at 37°C until mid-log phase (OD600 ~0.6)

• Inducing expression with IPTG (0.5-1 mM) at 18-20°C overnight to minimize inclusion body formation

For enhanced expression, codon optimization for E. coli may be necessary, as V. vulnificus has a different codon usage bias . Temperature reduction during induction is critical as it improves protein folding and solubility.

What purification strategies yield the highest purity and activity of V. vulnificus DTD?

A multi-step purification approach is recommended for obtaining highly pure and active DTD:

• Initial capture: Affinity chromatography using a His-tag (N or C-terminal) with Ni-NTA resin

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose)

• Polishing step: Size exclusion chromatography (Superdex 75/200)

Maintaining 5 mM DTT or 2 mM β-mercaptoethanol in all buffers is crucial to prevent oxidation of cysteine residues. Additionally, including 5% glycerol in the final buffer enhances protein stability during storage .

What quality control methods should be employed to verify the activity of purified recombinant DTD?

Several methods can be employed to verify the activity and quality of purified recombinant V. vulnificus DTD:

• Enzymatic Activity Assay: Measure the deacylation of D-Tyr-tRNA^Tyr^ using thin-layer chromatography (TLC)

  • Incubate 500 pM of DTD with 0.2 μM radiolabeled D-Tyr-tRNA^Tyr^ at 30°C

  • Sample at various time points and digest with S1 nuclease

  • Analyze by TLC using 100 mM ammonium chloride and 5% glacial acetic acid as mobile phase

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Thermal shift assay to determine stability (Tm)

  • Dynamic light scattering (DLS) to confirm monodispersity

• Substrate Specificity Verification:

  • Test activity against multiple D-aminoacyl-tRNAs

  • Verify lack of activity against L-aminoacyl-tRNAs

  • Assess activity on Gly-tRNA^Ala^ to confirm extended function

What is the catalytic mechanism of V. vulnificus DTD?

The catalytic mechanism of V. vulnificus DTD likely follows that elucidated for DTDs from other organisms. The mechanism involves:

• Substrate binding: The D-aminoacyl-tRNA is positioned in the active site with the ester bond accessible to the catalytic threonine

• Nucleophilic attack: The threonine residue acts as the main nucleophile, attacking the carbonyl carbon of the ester bond

• Transition state stabilization: Phenylalanine and glutamine residues stabilize the oxyanion hole

• Bond cleavage: The ester bond between the D-amino acid and tRNA is hydrolyzed

• Product release: The cleaved D-amino acid and free tRNA are released from the enzyme

Critically, unlike many hydrolytic enzymes, DTD does not rely on amino acid side chains for direct participation in catalysis. Instead, the enzyme creates a precise geometric arrangement that facilitates the hydrolysis reaction .

How does V. vulnificus DTD discriminate between D- and L-amino acids?

The enantioselectivity of V. vulnificus DTD likely depends on the presence of a Gly-Cis-Pro dipeptide, similar to that observed in Plasmodium falciparum DTD. This motif creates a chiral selection filter that:

• Accommodates the D-configuration of amino acids

• Sterically rejects L-amino acids due to clashes with the enzyme's active site

• Positions the scissile bond for optimal hydrolysis only when D-amino acids are present

The critical cross-subunit configuration creates an active site architecture that provides strict geometric constraints, allowing only D-amino acids to be properly positioned for hydrolysis. This mechanism ensures high specificity for D-aminoacyl-tRNAs while preventing hydrolysis of the biologically essential L-aminoacyl-tRNAs .

What are the kinetic parameters of V. vulnificus DTD compared to DTDs from other species?

While specific kinetic data for V. vulnificus DTD is not directly available in the search results, comparisons can be made based on studies of DTDs from other organisms:

V. vulnificus DTD would likely exhibit significantly higher activity (~1000-fold) on Gly-tRNA^Ala^ compared to Gly-tRNA^Gly^, similar to DTDs from other species . This substrate preference is attributed to the G3- U70 wobble base pair present in tRNA^Ala^, which acts as a positive determinant for DTD recognition.

What structural features distinguish V. vulnificus DTD from DTDs of other bacterial species?

Based on comparative analysis with other bacterial DTDs, V. vulnificus DTD likely shares core structural elements while potentially possessing unique features that may include:

• Conserved core architecture: A dimeric structure with each monomer adopting a fold similar to other bacterial DTDs

• Active site motif: Likely contains the highly conserved "SQFT" motif where threonine acts as the main nucleophile

• Species-specific variations: Potential adaptations in surface residues that might reflect the marine environment of V. vulnificus

• Thermostability modifications: As V. vulnificus thrives in warm coastal waters, its DTD may contain adaptations for enhanced thermostability compared to DTDs from mesophilic organisms

Genomic analysis of V. vulnificus strains reveals significant genetic diversity, with clinical and environmental isolates showing distinct genetic profiles . This diversity may extend to the dtd gene, potentially resulting in structural variations that could affect enzyme function or stability.

How does the G3- U70 recognition mechanism function in tRNA substrate selection?

The G3- U70 wobble base pair recognition mechanism is a fascinating aspect of DTD function that extends beyond simple chiral discrimination. This mechanism:

• Acts as a positive determinant for DTD recognition of tRNA^Ala^

• Results in significantly higher (~1000-fold) activity on Gly-tRNA^Ala^ compared to Gly-tRNA^Gly^

• Represents an unexpected tRNA-based code for DTD action

Experimental evidence demonstrated that transplanting G3- U70 into tRNA^Gly^ increased DTD activity by more than 10-fold compared to wild-type tRNA^Gly^. Importantly, substituting G3- C70 with another Watson-Crick pair (A3- U70) did not increase DTD activity, confirming the specificity for the G3- U70 wobble base pair .

This recognition mechanism has significant implications for understanding the evolution of the translation quality control system, as it suggests that DTD has adapted to recognize specific tRNA features beyond simply detecting the D-configuration of amino acids.

What insights can be gained from comparing V. vulnificus DTD with human DTD for potential drug development?

Comparing V. vulnificus DTD with human DTD offers several insights relevant to drug development:

• Structural differences: While both enzymes perform the same function, bacterial and human DTDs have evolved distinct structural features that could be exploited for selective inhibition

• Active site architecture: Differences in the configuration of the active site could allow for the design of inhibitors specific to bacterial DTDs

• Substrate preferences: Potential differences in substrate recognition and processing efficiency might be leveraged for selective targeting

The development of DTD inhibitors specific to V. vulnificus could represent a novel therapeutic approach, especially given the increasing concern about antimicrobial resistance. Since DTD is essential for preventing D-amino acid toxicity, its inhibition could potentially reduce V. vulnificus virulence or survival .

How does DTD contribute to V. vulnificus virulence and pathogenicity?

While the direct connection between DTD and V. vulnificus virulence has not been explicitly established in the search results, several hypotheses can be proposed based on our understanding of DTD function and V. vulnificus pathogenesis:

• Translational fidelity maintenance: By preventing D-amino acid incorporation, DTD ensures proper protein synthesis, which is essential for expressing virulence factors

• Stress adaptation: DTD may help V. vulnificus adapt to stressful conditions encountered during infection by maintaining translational accuracy under stress

• Response to host defense mechanisms: Host immune cells can produce D-amino acids as an antimicrobial strategy; DTD may help V. vulnificus resist this defense mechanism

V. vulnificus contains numerous virulence genes across multiple functional categories, including adherence, antiphagocytosis, chemotaxis/motility, iron uptake, and toxin production . The proper expression of these virulence factors likely depends on the translational quality control provided by DTD.

Is there a correlation between DTD sequence variations and the virulence of different V. vulnificus strains?

Genomic analysis of V. vulnificus strains has revealed significant genetic diversity between clinical and environmental isolates . While the search results don't explicitly address DTD sequence variations across strains, we can consider the following:

• Clinical isolates of V. vulnificus form relatively coherent phylogenetic groups, with 70% forming a monophyletic clade, suggesting conserved genetic lineages

• Environmental strains show greater phylogenetic dispersion, indicating higher genetic heterogeneity

• Sequence variations in DTD could potentially affect its activity, stability, or substrate specificity

The table below outlines potential correlations between strain characteristics and DTD variations:

A comprehensive analysis of DTD sequences across multiple V. vulnificus strains, correlated with virulence data, would be needed to establish definitive connections between DTD variations and pathogenicity.

Could DTD serve as a potential target for developing antimicrobial strategies against V. vulnificus?

DTD represents a promising target for antimicrobial development against V. vulnificus for several reasons:

• Essential function: DTD plays a crucial role in preventing D-amino acid toxicity, making it potentially essential for bacterial survival

• Unique mechanism: The distinct catalytic mechanism of DTD offers opportunities for specific inhibitor design

• Differences from human DTD: Structural and functional differences between bacterial and human DTDs could allow for selective targeting

Potential antimicrobial strategies targeting DTD could include:

• Development of small molecule inhibitors that specifically bind to the active site of bacterial DTDs

• Design of D-amino acid analogs that competitively inhibit DTD function

• Creation of tRNA mimics that bind to DTD but resist deacylation, thus sequestering the enzyme

These approaches could complement existing treatment strategies for V. vulnificus infections, which currently include antibiotics like doxycycline, ceftazidime, and ciprofloxacin .

What are the recommended assays for measuring DTD activity in vitro?

Several assays can be employed to measure DTD activity in vitro, each with specific advantages:

• Radiometric TLC-based assay:

  • Incubate DTD with radiolabeled D-Tyr-tRNA^Tyr^

  • Sample at various time points and digest with S1 nuclease

  • Separate products by thin-layer chromatography

  • Quantify using phosphorimaging

• Fluorescence-based assays:

  • Use fluorescently labeled tRNAs or amino acids

  • Monitor deacylation by changes in fluorescence intensity or anisotropy

  • Allows for real-time kinetic measurements

• Coupled enzyme assays:

  • Link DTD activity to a secondary reaction that produces a detectable signal

  • For example, couple amino acid release to oxidation by amino acid oxidase, detecting H₂O₂ production

When selecting an assay, researchers should consider the specific experimental goals, available equipment, and required throughput.

How can one generate and validate a V. vulnificus DTD knockout strain for functional studies?

Creating and validating a V. vulnificus DTD knockout strain involves several steps:

• Generation of the knockout construct:

  • Design primers to amplify regions flanking the dtd gene

  • Use SOE PCR (Splicing by Overlap Extension) to create a deletion construct

  • Clone the construct into a suicide vector (e.g., pDS132)

• Introduction into V. vulnificus:

  • Transform the construct into an appropriate E. coli donor strain (e.g., β2155)

  • Conjugate the plasmid into V. vulnificus

  • Select for integration using appropriate antibiotics (typically chloramphenicol)

  • Counter-select using sucrose to identify colonies that have undergone the second recombination event

• Validation of the knockout:

  • PCR verification of the deletion

  • RT-PCR to confirm absence of transcript

  • Western blot to verify absence of protein (requires specific antibodies)

  • Complementation studies to confirm phenotype is due to the deletion

• Phenotypic characterization:

  • Growth curve analysis under various conditions (different temperatures, salinities, D-amino acid concentrations)

  • D-amino acid tolerance tests

  • Translation fidelity assays

  • Virulence assessment in appropriate models

This approach has been successfully used to create gene knockouts in V. vulnificus, as demonstrated in the literature for other genes such as siaM .

What advanced structural biology techniques are most informative for studying DTD-substrate interactions?

Several advanced structural biology techniques can provide valuable insights into DTD-substrate interactions:

• X-ray crystallography:

  • Allows determination of high-resolution structures of DTD-substrate complexes

  • Substrate analogs like D-Tyr3AA can be used to capture enzyme-substrate interactions

  • Provides detailed information about the active site architecture and substrate binding

• NMR spectroscopy:

  • ¹⁵N-¹H TROSY experiments can detect chemical shift perturbations upon substrate binding

  • Can distinguish between binding to 2'-OH vs. 3'-OH of adenosine

  • Provides information about protein dynamics during catalysis

• Cryo-electron microscopy (Cryo-EM):

  • Allows visualization of larger complexes, potentially including DTD bound to full tRNA

  • Can capture different conformational states during the catalytic cycle

  • Does not require crystallization, which can be challenging for some complexes

• Molecular dynamics simulations:

  • Can model the dynamic behavior of DTD-substrate interactions over time

  • Helps identify transient interactions not captured by static structural methods

  • Allows investigation of the catalytic mechanism at atomic resolution

These techniques are complementary and can be combined to obtain a comprehensive understanding of DTD function. For example, X-ray structures provide a static view of the enzyme-substrate complex, while NMR and molecular dynamics provide insights into the dynamic aspects of catalysis.

What are the emerging research directions for understanding DTD function beyond traditional roles?

Recent discoveries have expanded our understanding of DTD function beyond its classical role in D-amino acid removal, opening several exciting research directions:

• Expanded substrate specificity:

  • Investigation of DTD's activity on Gly-tRNA^Ala^ reveals a role in correcting achiral amino acid mischarging

  • Exploration of other potential non-canonical substrates could further expand our understanding of DTD's quality control functions

• tRNA recognition mechanisms:

  • The discovery that the G3- U70 wobble base pair serves as a positive determinant for DTD suggests the existence of a tRNA-based code for DTD action

  • Further research into tRNA structural elements recognized by DTD could reveal additional specificity determinants

• Role in stress response:

  • Investigation of DTD function under various stress conditions relevant to V. vulnificus pathogenesis

  • Examination of potential regulatory mechanisms controlling DTD expression and activity during stress

• Evolutionary aspects:

  • Comparative analysis of DTD across different V. vulnificus strains and related Vibrio species

  • Investigation of how DTD has evolved to maintain translational fidelity while adapting to specific ecological niches

These research directions could significantly enhance our understanding of DTD's role in translational quality control and bacterial physiology.

How might DTD function be affected by environmental conditions relevant to V. vulnificus ecology?

V. vulnificus inhabits coastal waters and can cause infections through consumption of raw seafood or wound exposure to seawater . Understanding how environmental conditions affect DTD function could provide insights into bacterial adaptation and virulence:

• Temperature effects:

  • V. vulnificus proliferates in warm coastal waters (>20°C)

  • Higher temperatures could affect DTD stability, activity, and substrate specificity

  • Research could examine how DTD function changes across the temperature range encountered by V. vulnificus

• Salinity adaptation:

  • As V. vulnificus transitions between varying salinity environments, osmotic stress could impact protein synthesis

  • DTD activity may be modulated to maintain translational fidelity under different salt concentrations

  • Experiments comparing DTD function at different salinities could reveal adaptations to marine environments

• pH variations:

  • During infection, V. vulnificus encounters different pH environments

  • The pH optimum of DTD and its stability across pH ranges relevant to infection could affect bacterial fitness

  • Structure-function studies at different pH values could provide insights into DTD's role during infection

• Oxidative stress:

  • Host immune responses generate oxidative stress

  • DTD function under oxidative conditions could be critical for bacterial survival during infection

  • Investigation of potential redox-sensitive residues in DTD could reveal regulation mechanisms

Understanding these environmental effects could help explain V. vulnificus ecology and pathogenesis, potentially leading to new intervention strategies.

What computational approaches could advance our understanding of V. vulnificus DTD function and evolution?

Advanced computational approaches offer powerful tools for investigating DTD function and evolution:

• Homology modeling and molecular dynamics:

  • Creation of accurate structural models of V. vulnificus DTD based on solved structures from other organisms

  • Simulation of enzyme dynamics, substrate binding, and catalysis

  • Investigation of how sequence variations might affect enzyme function

• Phylogenetic analysis:

  • Comprehensive evolutionary analysis of DTD across V. vulnificus strains and related species

  • Correlation of DTD sequence variations with ecological niches and pathogenicity

  • Identification of conserved residues essential for function versus variable regions that may confer specific adaptations

• Genome-wide association studies (GWAS):

  • Analysis of correlations between DTD sequence variations and phenotypic traits across many V. vulnificus isolates

  • Identification of potential epistatic interactions between DTD and other genes

• Systems biology approaches:

  • Integration of DTD function into broader models of V. vulnificus metabolism and stress response

  • Prediction of the systemic effects of DTD perturbation

  • Identification of potential regulatory networks controlling DTD expression

• Machine learning applications:

  • Development of predictive models for DTD substrate specificity based on tRNA structural features

  • Identification of novel inhibitors through virtual screening and activity prediction

These computational approaches, combined with experimental validation, could significantly advance our understanding of DTD function and its role in V. vulnificus biology.

How could recombinant V. vulnificus DTD be utilized as a biotechnological tool?

Recombinant V. vulnificus DTD offers several potential biotechnological applications:

• D-amino acid detection system:

  • Development of DTD-based biosensors for detecting D-amino acids in biological samples

  • Application in quality control for pharmaceutical and food industries where D-amino acid content is relevant

• tRNA quality control in cell-free protein synthesis:

  • Addition of DTD to cell-free translation systems to improve protein synthesis fidelity

  • Removal of D-aminoacyl-tRNAs that might form during in vitro translation

• Chiral separation technology:

  • Exploitation of DTD's stereospecificity for developing enzymatic methods for separating D- and L-amino acids

  • Application in analytical chemistry and pharmaceutical production

• Research tool for studying translation:

  • Use of DTD to investigate the effects of D-amino acid incorporation on protein folding and function

  • Application in studies of translational quality control mechanisms

• Protein engineering platform:

  • Engineering DTD variants with altered substrate specificities

  • Development of DTD-based enzymatic cascades for biotechnological applications

The unique properties of V. vulnificus DTD, potentially including enhanced stability or activity under specific conditions, could make it particularly valuable for certain biotechnological applications.

What are the prospects for developing DTD inhibitors as novel antimicrobials against V. vulnificus?

The development of DTD inhibitors as antimicrobials against V. vulnificus presents both opportunities and challenges:

Opportunities:

• Novel target with essential function in maintaining translational fidelity

• Potential for selective toxicity based on structural differences between bacterial and human DTDs

• Possible synergy with existing antibiotics by compromising bacterial protein synthesis quality control

Challenges:

• Need for high selectivity to avoid affecting human DTD

• Requirement for cell permeability to reach the cytoplasmic target

• Potential for resistance development

Potential inhibitor development strategies:

• Structure-based design utilizing the known catalytic mechanism and active site architecture

• High-throughput screening of compound libraries against purified recombinant DTD

• Fragment-based drug discovery focusing on the substrate binding pocket

• Development of transition state analogs that bind tightly to the enzyme

Given the rising concern about antibiotic resistance and the severity of V. vulnificus infections, with mortality rates of approximately 20% , exploring novel targets like DTD represents a valuable approach to expanding our antimicrobial arsenal.

How could understanding of DTD function contribute to developing antimicrobial blue light therapy for V. vulnificus infections?

Recent research has explored antimicrobial blue light (aBL) therapy at 405 nm wavelength for treating V. vulnificus infections . Understanding DTD function could enhance this approach in several ways:

• Mechanistic synergy:

  • aBL generates reactive oxygen species (ROS) that damage bacterial cells

  • DTD inhibition could potentially sensitize V. vulnificus to oxidative stress by compromising protein quality control

  • Combined approaches targeting both DTD and ROS production could enhance antimicrobial efficacy

• Photosensitizer development:

  • Knowledge of DTD structure could inform the design of photosensitizers that specifically target V. vulnificus

  • DTD-binding molecules with photoreactive properties could deliver localized photodynamic effects

• Treatment optimization:

  • Understanding how DTD function changes under aBL exposure could help optimize treatment parameters

  • Investigation of potential adaptive responses involving DTD (e.g., upregulation) could inform treatment regimens to prevent resistance

• Biofilm considerations:

  • DTD's role in biofilm formation or maintenance could affect aBL therapy effectiveness

  • Combined approaches targeting both planktonic bacteria and biofilms might be necessary for effective treatment