Recombinant Vibrio vulnificus Glutamate 5-kinase (proB)

What is Vibrio vulnificus Glutamate 5-kinase and what is its role in bacterial metabolism?

Vibrio vulnificus Glutamate 5-kinase (proB, EC 2.7.2.11) is a key enzyme that catalyzes the first step in proline biosynthesis, converting glutamate to glutamate 5-phosphate using ATP. This enzyme (also known as gamma-glutamyl kinase or GK) is encoded by the proB gene (VV0858) in V. vulnificus strain YJ016 and consists of 379 amino acids . The enzyme plays a critical role in proline metabolism, which is particularly important for V. vulnificus during osmotic stress conditions. Research indicates that V. vulnificus cells accumulate higher levels of glutamate when challenged with NaCl and various hyperosmotic stresses, suggesting glutamate functions as a compatible solute in this pathogen .

How does the amino acid sequence of V. vulnificus Glutamate 5-kinase compare to homologs in other bacteria?

The V. vulnificus Glutamate 5-kinase (proB) full sequence (379 amino acids) is:

MTTNQQSVAA SQPKTIVVKL GTSVLTGGTL ALDRAHMVEL ARQCAELKKQ GHSVVVVSSG AIAAGREHLG YPALPNAMAS KQLLAAVGQS QLIQTWESLF ALYGIKIGQM LLTRADLEDR ERFLNARDTI NALVDNGIIP VVNENDAVAT SEIKVGDNDN LSALVGILCG ADKLLLLTDQ KGLYTADPRK DPNAELIKEV KVIDDTLRKI AGGSGTTLGT GGMATKLQAA DIARRAGIEV IIAAGRGQNV IFDALSPAPQ GTRFLPCEEA LENRKRWILA GPAASGDIVI DQGAVKAVVE KGSSLLAKGV TKVLGEFSRG EVVRVTDAQG HLVARGIASY SNQDMAKIAG KHSKDIISIL GYDYGSEVIH RDDMVVIQE

Comparative analysis with other bacterial Glutamate 5-kinases reveals conserved domains associated with substrate binding and catalytic activity. While the search results don't provide direct comparisons, researchers studying this enzyme should perform multiple sequence alignments using tools like Clustal Omega or MUSCLE to identify conserved regions across different bacterial species, which may provide insights into evolutionary relationships and functional conservation.

What are the optimal expression systems for recombinant V. vulnificus Glutamate 5-kinase production?

Multiple expression systems have been successfully used for the production of recombinant V. vulnificus Glutamate 5-kinase:

What purification strategies yield the highest purity and activity for recombinant V. vulnificus Glutamate 5-kinase?

While specific purification protocols for V. vulnificus Glutamate 5-kinase are not detailed in the search results, general methodological approaches for recombinant proteins with similar characteristics can be recommended:

• Affinity Chromatography: For tagged versions of the protein, use appropriate affinity resins (Ni-NTA for His-tagged proteins, streptavidin for Avi-tag biotinylated proteins).

• Ion Exchange Chromatography: Based on the theoretical pI of the protein calculated from its amino acid sequence.

• Size Exclusion Chromatography: As a polishing step to achieve >95% purity.

For optimal activity preservation:

• Use buffers containing stabilizing agents such as glycerol (10-20%)

• Include reducing agents like DTT or β-mercaptoethanol to prevent oxidation of cysteine residues

• Optimize salt concentration based on the enzyme's native environment (V. vulnificus is a halophilic bacterium)

• Consider including cofactors or substrate analogs for stability

The recommended final product should achieve >85% purity as verified by SDS-PAGE .

What are the established methods for measuring Glutamate 5-kinase activity in vitro?

While the search results don't provide specific assays for V. vulnificus Glutamate 5-kinase, standard enzymatic assays for this class of enzymes typically include:

• Coupled Enzyme Assay: Similar to methods used for AMP ligases in vulnibactin biosynthesis , measuring the formation of ADP or phosphate:

  • The reaction (Glutamate + ATP → Glutamate-5-phosphate + ADP) can be coupled to pyruvate kinase and lactate dehydrogenase reactions

  • Monitor NADH oxidation at 340 nm to indirectly measure kinase activity

• Direct Phosphate Detection:

  • Using colorimetric methods like malachite green to detect inorganic phosphate released in a coupled reaction with a phosphatase

  • Using pyrophosphatase to convert released pyrophosphate to phosphate for detection

• Mass Spectrometry:

  • Direct detection of the product (glutamate-5-phosphate) using LC-MS/MS

  • Quantification using isotopically labeled standards

Recommended assay conditions: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM ATP, 10 mM glutamate, at 30°C.

How does osmotic stress affect the expression and activity of V. vulnificus Glutamate 5-kinase?

Research on V. vulnificus indicates that cells accumulate higher levels of glutamate when challenged with NaCl and various hyperosmotic stresses . This suggests that Glutamate 5-kinase, as part of the proline biosynthesis pathway, may be regulated in response to osmotic conditions.

To study this experimentally:

• Expression Analysis:

  • Grow V. vulnificus in media with varying NaCl concentrations (0.5% to 3.5%)

  • Perform qRT-PCR to measure proB transcript levels

  • Use Western blotting to track protein expression levels

• Activity Measurements:

  • Isolate enzyme from cells grown under different osmotic conditions

  • Compare kinetic parameters (Km, Vmax) to determine if enzymatic properties change

  • Test enzyme activity in the presence of varying salt concentrations in vitro

• Computational Analysis:

  • Examine the promoter region of proB for regulatory elements related to osmotic stress

  • Look for transcription factor binding sites associated with osmotic stress response

This work could be connected to findings about putAP genes in V. vulnificus, which showed that proline metabolism is linked to osmotic stress adaptation .

How does Glutamate 5-kinase fit into the genome-scale metabolic network of V. vulnificus?

V. vulnificus metabolic network (VvuMBEL943) reconstruction includes 943 reactions and 765 metabolites, covering 673 genes . Within this network, Glutamate 5-kinase (proB) functions as a key enzyme in amino acid metabolism, specifically in the glutamate-to-proline biosynthesis pathway.

Researchers should consider:

This systems-level understanding can help position Glutamate 5-kinase within the broader context of V. vulnificus metabolism and potentially identify it as a drug target if it meets essentiality criteria.

What metabolomic approaches are suitable for tracing glutamate and proline metabolism in V. vulnificus?

For comprehensive analysis of glutamate and proline metabolism involving Glutamate 5-kinase:

• Isotope Labeling Studies:

  • Feed V. vulnificus with 13C-labeled glutamate or related precursors

  • Track isotope distribution using GC-MS or LC-MS/MS to determine metabolic flux

  • Calculate flux ratios through different branches of metabolism

• Targeted Metabolomics:

  • Focus on quantifying specific intermediates in the glutamate-proline pathway

  • Monitor changes in metabolite pools under different growth conditions or genetic modifications

  • Recommended metabolites to monitor: glutamate, glutamate-5-phosphate, glutamate-5-semialdehyde, pyrroline-5-carboxylate, and proline

• Metabolic Modeling:

  • Integrate experimental data with the VvuMBEL943 metabolic model

  • Simulate the effects of environmental changes or enzyme inhibition

  • Generate testable hypotheses about metabolic adaptation

How does Glutamate 5-kinase activity contribute to V. vulnificus virulence and survival in host environments?

Although the search results don't directly link Glutamate 5-kinase to virulence, we can hypothesize its role based on related findings:

• Osmotic Stress Adaptation:

  • V. vulnificus accumulates glutamate as a compatible solute during osmotic stress

  • Glutamate 5-kinase may help regulate this process by channeling glutamate into proline biosynthesis

  • This adaptation likely contributes to survival in varying salinity environments, including human tissues

• Connection to Quorum Sensing:

  • V. vulnificus uses quorum sensing molecules like cyclo(Phe-Pro) to modulate host immune responses

  • Since Glutamate 5-kinase is involved in proline metabolism, it may indirectly influence the production of proline-containing signaling molecules

• Potential Interaction with Iron Acquisition:

  • Iron acquisition through siderophores like vulnibactin is crucial for V. vulnificus virulence

  • Amino acid metabolism pathways may intersect with siderophore biosynthesis pathways under iron-limited conditions

Experimental approaches to test these hypotheses include:

• Creating proB knockout mutants and testing their virulence in animal models

• Comparing growth of wild-type and proB mutants under various stress conditions

• Measuring proline and glutamate levels in wild-type vs. mutant strains during infection

Can Glutamate 5-kinase be targeted for antimicrobial development against V. vulnificus?

The systems biological approach described for V. vulnificus drug targeting provides a framework for evaluating Glutamate 5-kinase as a potential antimicrobial target:

• Target Validation Criteria:

  • Determine if proB meets the "metabolite essentiality" concept, where inhibition would disrupt critical cell functions

  • Assess whether glutamate-5-phosphate is an essential metabolite in V. vulnificus

  • Compare with the five essential metabolites already identified in previous research

• Inhibitor Development Strategy:

  • Design chemical analogs of glutamate that could competitively inhibit Glutamate 5-kinase

  • Screen for compounds that interact with conserved domains in the enzyme

  • Test candidate inhibitors in whole-cell assays as described in metabolic network-based drug discovery

• Selectivity Considerations:

  • Compare sequence and structural differences between bacterial and human glutamate kinases

  • Target unique structural features of the bacterial enzyme

  • Design inhibitors that exploit differences in the active site

The advantage of this approach is that it integrates genomic information with metabolic modeling to identify targets that are essential for bacterial survival but have minimal impact on the host metabolism.

How can protein engineering be applied to modify the catalytic properties of V. vulnificus Glutamate 5-kinase?

Protein engineering of V. vulnificus Glutamate 5-kinase could focus on:

• Structure-Guided Mutagenesis:

  • Target residues in the ATP-binding site to alter kinetics

  • Modify substrate specificity by changing residues in the glutamate-binding pocket

  • Introduce mutations in regulatory domains to affect allosteric control

• Domain Swapping:

  • Exchange domains with homologous proteins from thermophilic organisms to increase stability

  • Create chimeric enzymes with domains from related kinases to alter function

  • Introduce domains that respond to different regulatory signals

• Tag Optimization:

  • Test various fusion strategies beyond the currently available tags

  • Evaluate the effect of tag position (N-terminal vs. C-terminal) on activity

  • Design linker sequences that minimize interference with catalytic function

Methodological considerations:

• Use computational modeling and molecular dynamics simulations to predict effects of mutations

• Employ high-throughput screening methods to evaluate variants

• Consider directed evolution approaches using error-prone PCR

What are the implications of genomic variation in the proB gene across different V. vulnificus strains?

Similar to the genetic variation observed in other V. vulnificus virulence factors like the rtxA1 gene , the proB gene may exhibit strain-specific variations that affect function:

• Comparative Genomic Analysis:

  • Sequence the proB gene from multiple clinical and environmental isolates

  • Identify single nucleotide polymorphisms (SNPs) and structural variations

  • Correlate genetic variations with differences in enzyme activity or regulation

• Evolutionary Considerations:

  • Examine whether the proB gene shows evidence of horizontal gene transfer

  • Assess selection pressure by calculating the ratio of synonymous to non-synonymous mutations

  • Determine if proB variants correlate with clinical vs. environmental isolates

• Functional Impact:

  • Express and characterize variants to determine differences in kinetic parameters

  • Test whether variants differ in their response to osmotic stress

  • Evaluate if strain-specific variations correlate with virulence potential

This research direction could reveal whether, like the rtxA1 gene that encodes MARTX toxins, the proB gene is "undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment" .

How can recombinant V. vulnificus Glutamate 5-kinase be used in high-throughput screening systems?

Researchers can utilize recombinant V. vulnificus Glutamate 5-kinase in high-throughput screening by:

• Assay Development:

  • Adapt enzymatic assays to microplate format using colorimetric or fluorescent readouts

  • Optimize reaction conditions for stability and sensitivity

  • Establish positive and negative controls for screening campaigns

• Screening Applications:

  • Screen chemical libraries for novel inhibitors as potential antimicrobials

  • Identify natural products that modulate enzyme activity

  • Test environmental samples for compounds that affect enzyme function

• Platform Integration:

  • Use biotinylated versions of the enzyme (with Avi-tag) for surface immobilization

  • Develop biosensor applications for detecting modulators of enzyme activity

  • Combine with microfluidic systems for increased throughput

What are the considerations for developing antibodies against V. vulnificus Glutamate 5-kinase for research applications?

For researchers developing antibodies against V. vulnificus Glutamate 5-kinase:

• Epitope Selection:

  • Choose unique regions that differentiate V. vulnificus Glutamate 5-kinase from homologs

  • Avoid highly conserved regions if specificity to V. vulnificus is required

  • Consider both linear and conformational epitopes

• Production Strategies:

  • Recombinant protein approach: Use purified full-length protein or specific domains

  • Synthetic peptide approach: Design peptides representing unique epitopes

  • Consider raising antibodies against different forms of the protein (native vs. denatured)

• Validation Methods:

  • Western blotting with recombinant protein and bacterial lysates

  • Immunoprecipitation to verify native protein recognition

  • Immunofluorescence to examine cellular localization

  • ELISA to quantify sensitivity and specificity

  • Testing cross-reactivity against other Vibrio species and related bacteria

• Applications:

  • Develop immunoassays for detecting V. vulnificus in environmental or clinical samples

  • Study protein expression levels under different conditions

  • Investigate protein-protein interactions through co-immunoprecipitation

  • Track protein localization during bacterial growth and stress response