Recombinant Vibrio vulnificus 8-amino-7-oxononanoate synthase (bioF)

What is 8-amino-7-oxononanoate synthase and what reaction does it catalyze?

8-amino-7-oxononanoate synthase (EC 2.3.1.47) is an enzyme that catalyzes the chemical reaction between 6-carboxyhexanoyl-CoA and L-alanine to produce 8-amino-7-oxononanoate, CoA, and CO₂ . This reaction represents a critical step in biotin metabolism. The enzyme employs pyridoxal phosphate as a cofactor for its catalytic activity . In the context of Vibrio vulnificus, this enzyme plays a role in the biotin biosynthetic pathway, which is essential for various cellular metabolic processes.

How is 8-amino-7-oxononanoate synthase classified biochemically?

This enzyme belongs to the family of transferases, specifically those acyltransferases that transfer groups other than aminoacyl groups . The systematic name of this enzyme class is 6-carboxyhexanoyl-CoA:L-alanine C-carboxyhexanoyltransferase (decarboxylating) . Alternative names used in scientific literature include 7-keto-8-aminopelargonic acid synthetase, 7-keto-8-aminopelargonic synthetase, and 8-amino-7-oxopelargonate synthase . The enzyme is indexed in various biochemical databases including BRENDA, KEGG, and MetaCyc, facilitating access to comprehensive information about its properties and functions.

What is the significance of studying bioF in Vibrio vulnificus?

Studying bioF in Vibrio vulnificus is significant because V. vulnificus is a deadly human pathogen that causes infections through seafood consumption or direct contact with wounds . Understanding the biochemical pathways, including biotin metabolism in which 8-amino-7-oxononanoate synthase participates, may provide insights into bacterial survival mechanisms and potential therapeutic targets. Additionally, the enzyme's role in biotin synthesis makes it metabolically important, as biotin is an essential cofactor for carboxylation, decarboxylation, and transcarboxylation reactions in various metabolic processes.

What expression systems are most effective for recombinant production of V. vulnificus bioF?

While the search results don't specifically address expression systems for V. vulnificus bioF, recombinant protein production typically begins with the construction of an expression vector that is introduced into a microbial host . For bacterial proteins like those from V. vulnificus, E. coli expression systems are often the first choice due to their simplicity, rapid growth, and high protein yields. The selection of an appropriate expression system should consider factors such as the protein's structural complexity, required post-translational modifications, and intended research applications.

How can researchers optimize the expression conditions for recombinant bioF?

Optimization of expression conditions is critical for obtaining adequate yields of functional protein. The traditional one-factor-at-a-time approach is inefficient and does not account for interactions between variables . Instead, Design of Experiments (DoE) approaches are recommended, allowing researchers to predict the effect of each factor and their interactions using a carefully selected small set of experiments . Key parameters to optimize include:

• Induction conditions (temperature, inducer concentration, timing)

• Media composition and supplements

• Host strain selection

• Codon optimization for the expression host

• Expression vector features (promoter, tags, fusion partners)

Several software packages are available to facilitate the choice of DoE approach, design of experiments, and analysis of results . This systematic optimization can significantly improve protein yield and quality while reducing time and resources spent.

What purification strategies are most effective for bioF while maintaining enzymatic activity?

While specific purification strategies for V. vulnificus bioF are not detailed in the search results, general principles of recombinant protein purification apply. Typically, a multi-step purification process is employed, beginning with cell lysis under conditions that preserve the protein's native structure. Affinity chromatography is often the initial purification step if the recombinant protein includes an affinity tag. This may be followed by ion exchange chromatography, size exclusion chromatography, or other techniques based on the protein's properties. Throughout the purification process, it's crucial to monitor enzyme activity to ensure that the native structure and function are maintained.

What analytical techniques are most appropriate for characterizing recombinant V. vulnificus bioF?

Characterization of recombinant bioF would typically include:

• Protein concentration determination (Bradford, BCA, absorbance at 280 nm)

• Purity assessment (SDS-PAGE, analytical size exclusion chromatography)

• Enzymatic activity assays (spectrophotometric methods, HPLC, mass spectrometry)

• Structural characterization (circular dichroism, thermal shift assays, limited proteolysis)

• Kinetic parameter determination (Km, Vmax, kcat for both substrates)

Each technique provides complementary information about the protein's properties and quality, allowing researchers to assess the success of their expression and purification protocols.

How can researchers assess the structural integrity of purified recombinant bioF?

Structural integrity can be assessed through multiple approaches:

• Circular dichroism spectroscopy to analyze secondary structure content

• Thermal denaturation studies to evaluate protein stability

• Size exclusion chromatography to confirm the protein exists in the expected oligomeric state

• Activity assays to verify that the enzyme retains its functional capabilities

• Mass spectrometry to confirm intact mass and identify potential modifications

For more detailed structural analysis, X-ray crystallography or cryo-electron microscopy might be employed, though these techniques require specialized equipment and expertise.

What approaches are recommended for crystallization of V. vulnificus bioF for structural studies?

Crystallization of recombinant proteins for structural studies typically begins with screening a wide range of conditions using commercially available crystallization screens. The optimization process can be enhanced using DoE approaches similar to those used for expression optimization . Factors affecting crystallization include:

• Protein concentration and purity

• Buffer composition (pH, salt concentration, additives)

• Temperature and crystallization method (hanging drop, sitting drop, etc.)

• Addition of ligands, substrates, or cofactors to stabilize specific conformations

The presence of the cofactor pyridoxal phosphate might be particularly important for obtaining crystals of bioF in a functionally relevant conformation.

How does V. vulnificus bioF activity compare with similar enzymes from other bacterial species?

Comparative analysis of bioF from V. vulnificus with homologous enzymes from other bacterial species can provide insights into evolutionary relationships and species-specific adaptations. Such comparison might include:

• Sequence alignment to identify conserved residues and domains

• Kinetic parameter comparison to detect functional differences

• Substrate specificity assessment to identify potential variations

• Structural comparison if 3D structures are available

These comparisons can help researchers understand the unique features of V. vulnificus bioF and potentially correlate them with the organism's ecological niche or pathogenicity.

Is there evidence for strain-specific variations in bioF among different V. vulnificus isolates?

While the search results don't specifically address bioF variations among V. vulnificus strains, they do indicate that V. vulnificus isolates from different sources (clinical vs. environmental) show variations in biochemical properties and genotypes . Clinical isolates tend to be less biochemically diverse than environmental isolates, particularly those from oysters . It would be reasonable to investigate whether bioF sequence or expression varies between different strains, especially between those with different virulence potentials or ecological niches.

How might bioF expression or function correlate with V. vulnificus virulence potential?

While direct evidence linking bioF to virulence is not provided in the search results, researchers have developed multiple methods to characterize clinical and environmental strains of V. vulnificus . These include real-time PCR methods targeting polymorphisms in the 16S rRNA, vcg, and pilF genes, as well as phenotypic methods like D-mannitol fermentation . A similar approach could be used to investigate potential correlations between bioF sequence variations, expression levels, or enzymatic properties and the virulence potential of different V. vulnificus strains.

How can Design of Experiments (DoE) be applied to optimize bioF enzyme activity assays?

Design of Experiments provides a systematic approach to optimize enzyme activity assays with minimal experimental runs . For bioF activity assays, researchers might consider the following factors:

• Buffer composition and pH

• Temperature and incubation time

• Substrate concentrations

• Cofactor (pyridoxal phosphate) concentration

• Enzyme concentration

A well-designed DoE approach would allow researchers to identify optimal conditions and understand the interaction effects between these factors. This is particularly important for enzyme assays, where multiple parameters can significantly affect the measured activity.

Table 1: Example DoE factors and levels for optimizing bioF enzyme activity assays

What controls and validation steps are essential when working with recombinant V. vulnificus bioF?

Essential controls and validation steps include:

• Negative controls (reaction mixture without enzyme, without substrate)

• Positive controls (if available, a well-characterized homologous enzyme)

• Validation of enzyme concentration determination

• Verification of substrate purity and stability

• Reproducibility assessment through technical and biological replicates

• Enzyme kinetic analysis to confirm Michaelis-Menten behavior

• Validation of analytical methods used to quantify reaction products

These controls help ensure that experimental results are reliable and that observed effects are truly attributable to the enzyme's activity.

How should researchers approach troubleshooting low activity of recombinant bioF?

When encountering low activity of recombinant bioF, researchers should systematically investigate potential causes:

• Protein misfolding or denaturation during expression or purification

• Incomplete incorporation of the pyridoxal phosphate cofactor

• Suboptimal assay conditions (pH, temperature, ionic strength)

• Substrate quality or concentration issues

• Presence of inhibitors or interfering compounds

• Formation of inactive oligomeric states

Addressing these factors systematically, potentially using DoE approaches to investigate multiple factors simultaneously, can help identify and resolve issues affecting enzyme activity.

How can site-directed mutagenesis be used to investigate structure-function relationships in V. vulnificus bioF?

Site-directed mutagenesis offers a powerful approach to probe the roles of specific amino acid residues in enzyme function. For bioF, researchers might target:

• Residues involved in pyridoxal phosphate binding

• Catalytic residues involved in the reaction mechanism

• Substrate binding site residues

• Residues potentially involved in oligomerization or structural stability

By creating specific mutants and characterizing their enzymatic properties, researchers can gain insights into the molecular mechanisms underlying bioF function.

What approaches could be used to investigate bioF regulation in V. vulnificus under different environmental conditions?

Investigation of bioF regulation might include:

• Transcriptional analysis (RT-qPCR, RNA-seq) to measure bioF expression under different conditions

• Reporter gene assays to identify regulatory elements controlling bioF expression

• Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the bioF promoter

• Proteomic analysis to quantify bioF protein levels

• Post-translational modification analysis to identify regulatory modifications

These approaches could help understand how V. vulnificus regulates biotin biosynthesis in response to environmental factors, potentially providing insights into its adaptation strategies.

How might bioF be exploited as a potential target for antimicrobial development against V. vulnificus?

As a key enzyme in biotin biosynthesis, bioF represents a potential target for antimicrobial development. Researchers might explore:

• High-throughput screening to identify inhibitors specific to V. vulnificus bioF

• Structure-based drug design if crystallographic data becomes available

• Evaluation of bioF essentiality through gene knockout studies

• Assessment of bioF inhibition effects on V. vulnificus virulence in infection models

• Comparative analysis with human biotin-related enzymes to ensure target specificity

Such research could potentially lead to new therapeutic strategies against this deadly pathogen.

How do clinical and environmental isolates of V. vulnificus differ, and what implications might this have for bioF studies?

Clinical and environmental isolates of V. vulnificus show distinct differences in biochemical properties and genotypes . Clinical isolates demonstrate less biochemical diversity than environmental isolates, particularly those from oysters . For bioF studies, these differences might suggest:

• Potential variations in bioF sequence, expression, or activity between clinical and environmental isolates

• Different regulatory mechanisms controlling bioF expression

• Possible correlation between bioF properties and virulence potential

Researchers studying bioF should consider these potential differences when selecting strains for their studies and when interpreting their results.

Table 2: Comparison of characteristics between clinical and environmental V. vulnificus isolates

What molecular typing methods could be applied to investigate bioF variations among V. vulnificus strains?

Based on the methods described for characterizing V. vulnificus strains , researchers could apply similar approaches to investigate bioF variations:

• PCR-based genotyping targeting polymorphisms in the bioF gene

• Real-time PCR assays for quantitative analysis

• Sequencing of the bioF gene and surrounding regulatory regions

• Restriction fragment length polymorphism (RFLP) analysis

• Whole genome sequencing and comparative genomics

These methods could help identify strain-specific variations in the bioF gene and potentially correlate them with other virulence or ecological markers.