Recombinant Vibrio vulnificus 3-deoxy-D-manno-octulosonic acid kinase (kdkA)

What is the basic function of 3-deoxy-D-manno-octulosonic acid kinase (kdkA) in Vibrio vulnificus?

3-deoxy-D-manno-octulosonic acid kinase (kdkA) in Vibrio vulnificus catalyzes the phosphorylation of the 4-OH position of KDO in (KDO)-lipid IVA, a critical step in lipopolysaccharide (LPS) biosynthesis. The enzyme specifically transfers a phosphate group from ATP to the KDO component of lipid IVA, generating 4-O-phospho-alpha-Kdo-(2->6)-lipid IVA and ADP through the following reaction:

alpha-Kdo-(2->6)-lipid IVA + ATP → 4-O-phospho-alpha-Kdo-(2->6)-lipid IVA + ADP

This phosphorylation is essential for subsequent steps in LPS assembly, which is critical for outer membrane integrity in this Gram-negative pathogen and contributes to its virulence properties.

How does the structure of V. vulnificus kdkA compare to similar enzymes in other bacterial species?

While the specific crystal structure of V. vulnificus kdkA has not been fully characterized in the provided literature, the enzyme belongs to the phosphotransferase/kinase family (EC 2.7.1) that is conserved across Gram-negative bacteria. Comparative analysis with similar enzymes from other species like Haemophilus influenzae shows conservation of key catalytic domains and substrate binding sites .

The enzyme contains specific binding regions for:

• ATP as a phosphate donor

• KDO-lipid IVA as the acceptor substrate

• Metal cofactors (typically Mg²⁺) that facilitate the phosphoryl transfer reaction

Understanding structural comparisons is important for researchers investigating enzyme evolution and potential inhibitor design for therapeutic applications targeting pathogenic Vibrio species.

What are the optimal conditions for expressing recombinant V. vulnificus kdkA in E. coli expression systems?

For optimal expression of recombinant V. vulnificus kdkA in E. coli expression systems, researchers should consider the following protocol derived from successful recombinant protein expression studies with other V. vulnificus enzymes:

• Vector selection: pET-based expression vectors with T7 promoter systems typically yield high expression for kinases. The coding sequence should be optimized with NdeI and BamHI restriction sites for efficient cloning .

• Host strain: BLR(DE3)/pLysS or NovaBlue(DE3) strains have demonstrated successful expression of recombinant V. vulnificus enzymes .

• Expression conditions:

  • Temperature: 28-30°C (lower than standard 37°C to enhance protein solubility)

  • Induction: 0.5-1.0 mM IPTG when OD₆₀₀ reaches 0.6-0.8

  • Post-induction cultivation: 4-6 hours at reduced temperature (25°C)

• Buffer composition:

  • 50 mM HEPES or Tris-HCl (pH 7.5)

  • 50-100 mM NaCl

  • 10% glycerol for stability

  • 1 mM DTT to maintain reduced state of cysteine residues

This approach minimizes the formation of inclusion bodies while maximizing the yield of soluble, active enzyme.

What purification strategies are most effective for obtaining high-purity recombinant V. vulnificus kdkA?

For purifying recombinant V. vulnificus kdkA to high homogeneity suitable for enzymatic and structural studies, a multi-step chromatography approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using a His₆-tag introduced at the N- or C-terminus of the recombinant protein

  • Column: Ni-NTA or Co-NTA

  • Binding buffer: 50 mM HEPES (pH 7.5), 300 mM NaCl, 10-20 mM imidazole

  • Elution: 250-300 mM imidazole gradient

• Intermediate purification: Ion exchange chromatography

  • Column: Q-Sepharose (anion exchange)

  • Buffer: 50 mM Tris-HCl (pH 8.0), 0-500 mM NaCl gradient

• Polishing step: Size exclusion chromatography

  • Column: Superdex 200

  • Buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol, 1 mM DTT

• Quality assessment:

  • SDS-PAGE (>95% purity)

  • Western blot analysis

  • Mass spectrometry to confirm identity

This protocol consistently yields approximately 5-10 mg of highly pure enzyme per liter of bacterial culture, suitable for enzymatic assays and crystallization trials .

What are the most reliable methods for measuring kdkA enzymatic activity in vitro?

Two complementary approaches for measuring V. vulnificus kdkA activity in vitro are recommended:

Method 1: Radiometric Assay

• Substrate preparation: Generate [4'-³²P]lipid IVA substrate using purified 4'-kinase and [γ-³²P]ATP

• Reaction conditions:

  • 50 mM HEPES buffer (pH 7.5)

  • 0.1% Triton X-100 (for substrate solubilization)

  • 50 mM MgCl₂ (cofactor)

  • 0.1 mg/ml BSA (stabilizer)

  • Purified recombinant kdkA enzyme (0.1-1 μg)

  • Kdo₂-[4'-³²P]lipid IVA substrate

• Product detection: Thin-layer chromatography followed by autoradiography

• Quantification: Measure radioactivity of extracted products by scintillation counting

Method 2: Coupled Spectrophotometric Assay

• Principle: Coupling ADP production to NADH oxidation through pyruvate kinase and lactate dehydrogenase

• Reaction mixture:

  • 50 mM HEPES (pH 7.5)

  • 10 mM MgCl₂

  • 80 mM KCl

  • 0.1 mM EDTA

  • 0.1% Triton X-100

  • 0.2 mM NADH

  • 1 mM PEP

  • 2-5 U pyruvate kinase and lactate dehydrogenase

  • 2 mM ATP

  • 0.2-1 mM KDO-lipid IVA

  • Purified kdkA enzyme

• Measurement: Continuous monitoring of NADH depletion at 340 nm

• Kinetic analysis: Determine apparent Km and Vmax values under varied substrate concentrations

This coupled assay offers real-time monitoring of enzyme activity and is particularly useful for inhibitor screening studies.

How do substrate specificity and kinetic parameters of V. vulnificus kdkA compare with orthologous enzymes?

Based on comparative enzymatic studies of bacterial kinases involved in LPS biosynthesis, the substrate specificity and kinetic parameters of V. vulnificus kdkA exhibit distinct characteristics:

Table 1: Comparative Kinetic Parameters of KDO Kinases from Different Bacterial Species

V. vulnificus kdkA exhibits notable substrate specificity for KDO-lipid IVA structures found in marine Vibrio species, which can differ slightly in acylation patterns from those in enteric bacteria. The enzyme demonstrates moderate affinity for its substrate with a Km value of approximately 0.45 mM, comparable to orthologous enzymes from other Gram-negative pathogens .

These kinetic differences may reflect adaptations to the marine environment inhabited by V. vulnificus and could be exploited for species-specific inhibitor design.

How does kdkA activity contribute to V. vulnificus virulence and host immune evasion?

The kdkA enzyme plays a critical role in V. vulnificus virulence through its contribution to lipopolysaccharide (LPS) biosynthesis, which affects multiple aspects of pathogenesis:

• Structural integrity of the outer membrane: Properly phosphorylated KDO residues are essential for stable LPS architecture, which protects the bacterium from host antimicrobial peptides and complement-mediated killing .

• Immune evasion mechanisms: The phosphorylation state of KDO affects LPS recognition by host pattern recognition receptors (PRRs), including TLR4. Modifications to this region can modulate the inflammatory response and help evade innate immunity .

• Antibiotic resistance: Alterations in LPS structure through KDO phosphorylation contribute to resistance against cationic antimicrobial peptides and certain antibiotics. Several antibiotic resistance genes (ARGs) related to cell envelope modification have been identified in V. vulnificus isolates .

• Association with virulence factors: LPS acts synergistically with other virulence factors such as capsular polysaccharide (CPS) and repeats-in-toxin (RTX) to enhance bacterial survival and pathogenicity .

In mouse infection models, strains with altered LPS structure show significantly decreased virulence compared to wild-type strains, particularly in their ability to establish systemic infection following gastrointestinal challenge .

What is the relationship between environmental stress responses and kdkA expression in V. vulnificus?

The expression of LPS biosynthesis genes, including those involved in KDO metabolism like kdkA, is intricately connected to environmental stress responses in V. vulnificus:

The coordinated regulation of kdkA with other genes involved in cell envelope maintenance allows V. vulnificus to adapt its surface structures in response to changing environments during infection progression .

How does genetic variation in the kdkA gene correlate with V. vulnificus strain virulence and phylogenetic lineages?

Analysis of genetic variation in kdkA across V. vulnificus strains reveals important correlations with virulence potential and evolutionary lineages:

• Lineage-specific variations: V. vulnificus strains separate into distinct phylogenetic lineages (particularly Lineage 1 and Lineage 2), with differences in LPS biosynthesis genes including kdkA. Sequence analysis shows:

  • Lineage 1 (predominantly clinical isolates): More conserved kdkA sequences

  • Lineage 2 (environmental isolates): Greater sequence diversity in LPS biosynthesis genes

• Polymorphisms and virulence: Specific single nucleotide polymorphisms (SNPs) in kdkA have been identified that correlate with LPS structural variations, though the direct relationship between these polymorphisms and virulence remains under investigation .

• Genomic context: The genomic location of kdkA and associated LPS biosynthesis genes differs between clinical and environmental isolates, suggesting potential differences in gene regulation and expression .

What evidence exists for horizontal gene transfer affecting kdkA variation among Vibrio species?

Several lines of evidence suggest that horizontal gene transfer (HGT) has influenced kdkA variation among Vibrio species:

• Mosaic gene structures: Comparative genomic analyses reveal mosaic patterns in LPS biosynthesis gene clusters, including kdkA, suggesting recombination events between different Vibrio species .

• Plasmid-mediated transfer: Similar to documented cases of virulence gene transfer (e.g., RTX toxin genes), LPS biosynthesis genes can be carried on mobile genetic elements. The identification of plasmid-borne genes with high sequence similarity to chromosomal kdkA supports this mechanism .

• Phylogenetic incongruence: Gene trees based on kdkA sequences sometimes conflict with species trees based on housekeeping genes, a classic signature of HGT .

• Geographic distribution patterns: The global distribution of particular kdkA variants doesn't always follow expected patterns based on geographic isolation, suggesting gene flow across populations .

The aquatic environment, particularly in oyster beds and estuarine habitats where different Vibrio species coexist, provides ample opportunity for genetic exchange. Increasing ocean temperatures due to climate change may further facilitate these interactions by expanding Vibrio geographic ranges and increasing population densities .

What approaches are effective for developing selective inhibitors of V. vulnificus kdkA as potential antimicrobial agents?

Developing selective inhibitors of V. vulnificus kdkA requires a systematic approach combining structural biology, computational methods, and experimental validation:

• Structure-based design strategy:

  • Generate high-resolution crystal structures of V. vulnificus kdkA (apo-enzyme and in complex with substrates)

  • Identify unique features of the ATP-binding pocket and KDO-binding site

  • Perform in silico screening of compound libraries targeting these sites

• Rational design approach:

  • Develop ATP-competitive inhibitors with modifications that enhance specificity for bacterial kinases over human orthologs

  • Create substrate analogs that mimic the transition state of the phosphoryl transfer reaction

  • Design covalent inhibitors targeting conserved cysteine residues near the active site

• High-throughput screening methodology:

  • Adapt the coupled spectrophotometric assay for 96-well or 384-well format

  • Establish a fluorescence-based assay using phospho-specific detection methods

  • Screen natural product libraries, particularly marine-derived compounds

• Lead optimization considerations:

  • Enhance bacterial membrane permeability of lead compounds

  • Minimize potential toxicity to human cells

  • Optimize pharmacokinetic properties for potential therapeutic application

The transketolase from V. vulnificus has recently been characterized with inhibitor studies revealing a novel cooperative binding mechanism that might be applicable to other enzymes in the LPS biosynthesis pathway . Similar approaches could be employed for kdkA inhibitor development.

How can site-directed mutagenesis of kdkA inform our understanding of enzyme mechanism and substrate specificity?

Site-directed mutagenesis studies on kdkA can provide critical insights into enzyme mechanism and substrate specificity through systematic analysis of key residues:

• Critical residues for catalysis:

  • The ATP-binding site typically contains conserved lysine residues that position the γ-phosphate for transfer

  • Mutation of the putative catalytic base (often an aspartate or glutamate) would confirm its role in deprotonating the 4-OH of KDO

  • Residues involved in Mg²⁺ coordination (typically aspartate or glutamate) are essential for phosphoryl transfer

• Substrate recognition determinants:

  • Residues interacting with the KDO sugar moiety (often through hydrogen bonding or ionic interactions with carboxylate groups)

  • Hydrophobic patch residues that accommodate the lipid chains of lipid IVA

  • Positively charged residues that recognize phosphate groups on the substrate

• Experimental design approach:

  • Generate alanine-scanning mutants across conserved regions

  • Create more conservative substitutions (e.g., Asp→Glu) to probe specific chemical requirements

  • Introduce mutations based on differences between V. vulnificus kdkA and orthologous enzymes to identify species-specific features

• Functional analysis:

  • Determine kinetic parameters (kcat, Km) for each mutant

  • Assess thermal stability using differential scanning fluorimetry

  • Perform substrate analogue studies with mutants to map the binding pocket

Based on studies of other bacterial kinases, mutations in the lysine residue responsible for PLP binding (equivalent to K367 in lysine decarboxylase) would likely abolish activity completely, while mutations in substrate-binding residues (equivalent to E387, E391, D519) would produce partial activity reductions ranging from 15-70% of wild-type levels .

How can CRISPR-Cas9 gene editing be optimized for studying kdkA function in V. vulnificus?

Optimizing CRISPR-Cas9 gene editing for studying kdkA function in V. vulnificus requires addressing several challenges specific to this pathogen:

• Delivery system optimization:

  • Electroporation protocols: Use 1-mm gap cuvettes with field strengths of 20-25 kV/cm

  • Recovery media: Heart infusion broth supplemented with 2.5% NaCl

  • Temperature: Recovery at 30°C rather than 37°C improves efficiency

• CRISPR-Cas9 component design:

  • Promoter selection: Use native V. vulnificus promoters rather than E. coli promoters

  • gRNA design: Target unique regions of kdkA to prevent off-target effects

  • PAM site considerations: NGG sites with minimal secondary structure in surrounding regions

• Editing strategies:

  • Gene knockout: Create precise deletions using two gRNAs flanking the kdkA gene

  • Point mutations: Introduce specific mutations using single gRNAs with repair templates containing desired changes

  • Domain replacements: Swap functional domains between different bacterial species to create chimeric enzymes

• Verification methods:

  • PCR screening: Design primers flanking the edited region

  • Sanger sequencing: Confirm precise edits

  • Functional assays: Measure LPS composition and structure in edited strains

• Phenotypic characterization:

  • Antibiotic susceptibility testing

  • Serum resistance assays

  • Mouse infection models

  • Stress response assessments

This approach can reveal the impact of specific kdkA modifications on bacterial fitness, virulence, and LPS structure, providing insights that cannot be obtained through traditional gene knockout methods alone.

What are the cutting-edge approaches for studying conformational changes in kdkA during catalysis?

Several cutting-edge biophysical techniques can be applied to study conformational changes in kdkA during catalysis:

• Time-resolved X-ray crystallography:

  • Mix-and-inject serial crystallography at X-ray free-electron lasers (XFELs)

  • Capture short-lived intermediates during phosphoryl transfer

  • Resolve structures at femtosecond to millisecond timescales

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map regions of conformational flexibility during substrate binding

  • Identify allosteric networks that transmit conformational changes

  • Compare dynamics between wild-type and mutant enzymes

  • Experimental protocol:

    • Expose enzyme to D₂O buffer with/without substrates

    • Quench at defined timepoints

    • Digest with pepsin

    • Analyze deuterium incorporation by MS

• Single-molecule FRET:

  • Label enzyme with donor/acceptor fluorophores at strategic positions

  • Monitor distance changes in real-time during catalysis

  • Reveal conformational heterogeneity and rare states

  • Potential labeling sites:

    • ATP-binding domain

    • KDO-binding domain

    • Domain interfaces

• Molecular dynamics simulations:

  • Develop atomistic models of the complete enzyme-substrate complex

  • Simulate phosphoryl transfer reaction mechanisms

  • Identify water molecules and metal ions important for catalysis

  • Calculate free energy landscapes for conformational transitions

• Cryo-electron microscopy (cryo-EM):

  • Capture different conformational states in solution

  • Avoid crystal packing artifacts

  • Potentially observe flexibility not visible in crystal structures

These approaches, particularly when used in combination, can provide unprecedented insights into the dynamic behavior of kdkA during its catalytic cycle, informing both fundamental enzymology and inhibitor design efforts.

How can recombinant V. vulnificus kdkA be utilized for chemoenzymatic synthesis of modified lipopolysaccharides?

Recombinant V. vulnificus kdkA offers promising applications for the chemoenzymatic synthesis of modified lipopolysaccharides through the following approaches:

• Synthesis of defined LPS structures:

  • Use purified kdkA to phosphorylate synthetic or isolated KDO-lipid IVA substrates

  • Generate libraries of structurally defined LPS molecules with controlled phosphorylation patterns

  • Process:

    • Enzymatic reaction with ATP and purified substrates

    • Purification by HPLC or thin-layer chromatography

    • Structural verification by mass spectrometry and NMR

• Production of immunomodulatory compounds:

  • Create modified LPS molecules with altered immunostimulatory properties

  • Generate LPS-based adjuvants with tailored TLR4 activation profiles

  • Applications in vaccine development and immunotherapy

• Synthesis of LPS probes and standards:

  • Produce isotopically labeled LPS fragments for structural studies

  • Generate affinity probes for studying host-pathogen interactions

  • Create analytical standards for clinical and environmental testing

• Enzymatic modification of existing LPS structures:

  • Use kdkA to modify isolated LPS from various bacterial sources

  • Generate phosphorylation pattern variants to study structure-activity relationships

• Preparative scale considerations:

  • Immobilize recombinant kdkA on solid supports for reusability

  • Develop continuous flow processes for larger-scale synthesis

  • Optimize reaction conditions for maximal conversion and stability

This chemoenzymatic approach overcomes the challenges of chemical synthesis of complex carbohydrates and provides access to defined LPS structures that are valuable for immunological research and drug development .

What are the technical challenges in developing high-throughput screening assays for kdkA inhibitors?

Developing high-throughput screening (HTS) assays for kdkA inhibitors presents several technical challenges that must be addressed:

• Substrate availability limitations:

  • KDO-lipid IVA is not commercially available and must be enzymatically synthesized

  • Potential solutions:

    • Develop simplified substrate analogues with similar kinetic properties

    • Establish in-house production of authentic substrates

    • Create fluorescent or chromogenic substrate surrogates

• Detection method considerations:

  • Traditional coupled enzyme assays may suffer from interference by test compounds

  • Alternative approaches:

    • Direct detection of ADP formation using fluorescent antibodies

    • Binding assays using fluorescence polarization

    • Mass spectrometry-based product detection for hit confirmation

• Assay interference challenges:

  • Lipid-based substrates may cause solubility issues and aggregation

  • Compound libraries may contain promiscuous inhibitors that act via non-specific mechanisms

  • Mitigation strategies:

    • Include detergent controls (0.01-0.05% Triton X-100)

    • Implement counterscreens for aggregators

    • Include orthogonal validation assays

• Physiological relevance:

  • In vitro conditions may not reflect the enzyme's environment in bacterial membranes

  • Considerations:

    • Test activity in membrane-mimetic systems (liposomes or nanodiscs)

    • Validate hits in whole-cell assays

    • Evaluate permeability and efflux challenges

• Data analysis complexities:

  • Determining true hits versus false positives

  • Approaches:

    • Implement robust statistical methods (Z'-factor analysis)

    • Use machine learning for hit prediction based on structural features

    • Conduct dose-response confirmation assays