Recombinant Salmonella paratyphi B Lipoyl synthase (lipA)

What is Lipoyl synthase (lipA) and what is its function in Salmonella paratyphi B?

Lipoyl synthase (lipA) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. In Salmonella paratyphi B, as in other organisms, lipA contains two [4Fe-4S] centers: a reducing cluster and an auxiliary cluster. These clusters promote radical formation and sulfur transfer, respectively, which are essential for the enzyme's catalytic function . The enzyme is crucial for metabolism as it creates lipoic acid, an essential cofactor for various enzyme complexes involved in oxidative metabolism. The full protein sequence of Salmonella paratyphi B lipA consists of 321 amino acids .

How does Salmonella paratyphi B lipA structurally compare to lipA from other organisms?

Salmonella paratyphi B lipA shares structural similarities with other bacterial lipA enzymes, particularly in conserved domains related to its catalytic function. Unlike human LIAS, where most mechanistic information has been derived from prokaryotic enzymes, Salmonella paratyphi B lipA has specific characteristics that make it valuable for comparative studies. The protein contains critical conserved cysteine residues that coordinate the [4Fe-4S] clusters . While the broad structure remains conserved across species, species-specific variations exist in substrate specificity and cluster assembly mechanisms. The full sequence provided in the product data sheet reveals key motifs characteristic of the radical SAM enzyme family .

What are the optimal storage and handling conditions for recombinant Salmonella paratyphi B lipA?

For optimal preservation of recombinant Salmonella paratyphi B lipA activity, the protein should be stored at -20°C, and for extended storage, conservation at -20°C or -80°C is recommended. Repeated freezing and thawing should be avoided as it can compromise protein integrity and activity. For working aliquots, storage at 4°C for up to one week is advised . Prior to opening, vials should be briefly centrifuged to bring contents to the bottom. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C . The default recommended final concentration of glycerol is 50%. The shelf life in liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form can be stored for 12 months at the same temperatures .

What are the key differences in iron-sulfur cluster assembly and function between Salmonella paratyphi B lipA and human LIAS?

The iron-sulfur cluster assembly mechanisms in Salmonella paratyphi B lipA and human LIAS exhibit important differences that impact experimental design considerations. Human LIAS studies have revealed that [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 are capable of reconstituting human LIAS, enabling complete product turnover as monitored via LC-MS assays . In contrast, bacterial lipA homologs, including Salmonella paratyphi B lipA, may utilize different cluster donor systems reflecting their unique cellular environments.

Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives lacking the ability to bind one or the other of LIAS's two [4Fe-4S] clusters suggest a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center . This ordering may be conserved in Salmonella paratyphi B lipA, but specific experimental verification is needed. When designing experiments to study lipA function, researchers should consider these differences in Fe-S cluster trafficking between bacterial and human systems, as they may influence reconstitution protocols and activity assays .

How can recombinant Salmonella paratyphi B lipA be used as a model system for investigating radical SAM enzyme mechanisms?

Recombinant Salmonella paratyphi B lipA serves as an excellent model system for investigating radical SAM enzyme mechanisms due to its well-characterized dual [4Fe-4S] cluster system. To effectively utilize this protein as a mechanistic model, researchers should:

• Design experiments that specifically probe the sequential action of the two [4Fe-4S] clusters

• Utilize spectroscopic techniques such as EPR to monitor radical formation

• Employ isotope labeling to track sulfur insertion into target substrates

The reducing cluster generates a 5'-deoxyadenosyl radical through reductive cleavage of S-adenosylmethionine, while the auxiliary cluster serves as the source of sulfur atoms for insertion into the substrate . By comparing wild-type and mutant forms of the enzyme with substitutions in key cysteine residues that coordinate either cluster, researchers can dissect the individual contributions of each cluster to catalysis. This approach has been successful with human LIAS and could be applied to Salmonella paratyphi B lipA to reveal conservation or divergence in radical SAM enzyme mechanisms .

What experimental approaches can resolve contradictory data on lipA activity in different buffer conditions?

Resolving contradictory data on lipA activity across different buffer conditions requires a systematic approach that accounts for multiple variables affecting enzyme function. When encountering contradictory results, researchers should:

• Standardize protein preparation methods to ensure consistent [4Fe-4S] cluster incorporation

• Evaluate buffer components for potential interference with iron-sulfur clusters

• Establish a matrix experimental design testing:

  • pH ranges (typically 7.0-8.5)

  • Ionic strength variations (50-300 mM)

  • Reducing agent concentrations (DTT, 2-mercaptoethanol)

  • Oxygen exposure during manipulation

Table 1: Buffer Optimization Matrix for Salmonella paratyphi B lipA Activity

The stability of iron-sulfur clusters is particularly sensitive to oxidation, and activity assays should be performed under anaerobic or low-oxygen conditions whenever possible . Additionally, the presence of specific metal ions can affect cluster stability and enzyme activity, necessitating careful control of metal contaminants in all buffer components .

What is the recommended protocol for reconstituting the iron-sulfur clusters in recombinant Salmonella paratyphi B lipA?

Reconstituting iron-sulfur clusters in recombinant Salmonella paratyphi B lipA requires careful attention to anaerobic technique and reagent quality. Based on studies with related proteins, the following protocol is recommended:

• Prepare all solutions anaerobically by sparging with nitrogen or argon

• In an anaerobic chamber, combine:

  • Purified apo-lipA protein (typically 50-100 μM)

  • Ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)₂) (10-fold molar excess)

  • Sodium sulfide (Na₂S) (10-fold molar excess)

  • DTT or β-mercaptoethanol (5 mM)

  • Buffer (typically 50 mM Tris-HCl, pH 8.0, 150 mM NaCl)

• Incubate the mixture at 4°C for 3-4 hours with gentle rotation

• Remove excess iron and sulfide by dialysis or gel filtration

• Verify cluster incorporation by UV-visible spectroscopy (characteristic absorption at ~410 nm)

For more efficient and physiologically relevant reconstitution, bacterial iron-sulfur cluster assembly proteins can be employed. While studies on human LIAS have shown that [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 can reconstitute the enzyme , the specific bacterial homologs capable of reconstituting Salmonella paratyphi B lipA have not been definitively identified. Researchers should consider exploring the efficiency of various bacterial Fe-S cluster donor proteins in reconstituting lipA activity, which could provide insights into the Fe-S cluster assembly pathways in Salmonella paratyphi B.

How can the enzymatic activity of recombinant Salmonella paratyphi B lipA be accurately measured?

Accurate measurement of enzymatic activity for recombinant Salmonella paratyphi B lipA requires specialized techniques that account for both radical SAM chemistry and the unique dual-cluster system. The following methodological approach is recommended:

• Sample preparation:

  • Use freshly reconstituted enzyme containing both [4Fe-4S] clusters

  • Prepare reaction components anaerobically

  • Include S-adenosylmethionine (SAM), substrate (typically octanoyl-E2 or octanoyl-H protein), dithionite (as electron donor), and appropriate buffer

• Activity assays:

  • HPLC or LC-MS analysis: Monitor the conversion of octanoyl substrates to lipoyl products by separating and quantifying reaction components

  • Radiolabeling: Use ³⁵S-labeled substrates to track sulfur incorporation

  • Electron paramagnetic resonance (EPR): Detect radical intermediates formed during catalysis

• Controls and validation:

  • Run parallel reactions with heat-inactivated enzyme

  • Include metal chelators in control reactions to confirm iron-dependency

  • Verify product identity by mass spectrometry

Research with human LIAS has employed liquid chromatography-mass spectrometry (LC-MS) assays to monitor complete product turnover , which could be adapted for Salmonella paratyphi B lipA. Confirmation of proper [4Fe-4S] cluster incorporation is essential for meaningful activity measurements, as partially reconstituted enzyme or enzyme with damaged clusters will show significantly reduced activity.

What considerations are important when using site-directed mutagenesis to study functional domains of Salmonella paratyphi B lipA?

Site-directed mutagenesis of Salmonella paratyphi B lipA provides valuable insights into structure-function relationships but requires careful consideration of several factors:

• Target selection:

  • Conserved cysteine residues that coordinate [4Fe-4S] clusters

  • Residues implicated in SAM binding

  • Residues involved in substrate recognition

  • Key catalytic residues based on sequence alignment with characterized lipA enzymes

• Mutagenesis strategy:

  • Conservative substitutions (e.g., Cys to Ser) to maintain similar structural properties

  • Complete functional knockouts (e.g., Cys to Ala) to eliminate specific functions

  • Introduction of non-canonical amino acids to probe electronic requirements

• Functional assessment:

  • Differential effects on the two [4Fe-4S] clusters

  • Specific impact on SAM binding versus substrate binding

  • Effects on protein stability versus catalytic activity

Drawing from studies on human LIAS, EPR analysis of substituted derivatives that lack the ability to bind one or the other of the two [4Fe-4S] clusters can reveal the order of cluster assembly and the specific roles of each cluster . When interpreting mutagenesis results, it's important to distinguish between direct effects on catalysis and indirect effects caused by structural perturbations or impaired cluster assembly.

How can recombinant Salmonella paratyphi B lipA be utilized in vaccine development research?

Recombinant Salmonella paratyphi B lipA presents opportunities for vaccine development research through multiple avenues:

• As a component in subunit vaccines:

  • The unique antigenic properties of lipA could be incorporated into vaccine formulations targeting Salmonella infections

  • Research should focus on epitope mapping to identify immunogenic regions specific to the pathogen

• As part of attenuated live vaccine vectors:

  • Salmonella enterica has shown promise as a delivery system for heterologous molecules in cancer therapy and could be adapted for vaccine applications

  • Recombinant lipA could be modified to enhance immunogenicity or serve as a fusion partner for other antigens

• For studying host-pathogen interactions:

  • lipA's role in bacterial metabolism makes it relevant for understanding Salmonella survival in host environments

  • Researchers can investigate how lipA activity affects bacterial fitness during infection

Recent work has demonstrated that Salmonella Paratyphi A O-antigen glycoconjugate vaccines can induce functional immune responses regardless of O-antigen structural variations . Similar approaches could be explored with Salmonella paratyphi B antigens, potentially including modified forms of lipA. When designing such studies, researchers should consider that formaldehyde-fixed Salmonella paratyphi B has been shown to function as a polyclonal activator of human peripheral blood B cells, which could influence immune responses to vaccine candidates .

What comparative analyses between bacterial and human lipoyl synthases yield insights for antimicrobial development?

Comparative analyses between Salmonella paratyphi B lipA and human LIAS can provide valuable insights for antimicrobial development through several approaches:

• Structural comparisons:

  • Identify unique features of bacterial lipA that can be targeted without affecting human LIAS

  • Focus on differences in substrate binding pockets and active site architecture

• Mechanistic distinctions:

  • Explore differences in iron-sulfur cluster assembly and stability

  • Investigate variations in reaction kinetics and intermediate formation

• Inhibitor development strategy:

  • Design inhibitors that selectively target bacterial lipA based on identified differences

  • Test cross-reactivity against human LIAS to ensure specificity

Studies with human LIAS have revealed specific pathways for Fe-S cluster trafficking and assembly that may differ from bacterial systems . These differences could be exploited to develop inhibitors that selectively block bacterial lipoic acid biosynthesis without interfering with human metabolism. Additionally, the essential nature of lipoic acid for bacterial growth makes lipA an attractive target for antimicrobial development, especially for pathogens that rely on de novo lipoic acid synthesis rather than scavenging from the host.

How should researchers interpret spectroscopic data from [4Fe-4S] cluster reconstitution experiments?

Interpreting spectroscopic data from [4Fe-4S] cluster reconstitution experiments requires careful consideration of multiple spectral features and their biological significance:

• UV-visible spectroscopy:

  • Characteristic absorbance at ~410 nm indicates [4Fe-4S] cluster formation

  • Calculate the ratio of A410/A280 to estimate cluster incorporation per protein molecule

  • Expected ratios for fully reconstituted lipA (with two clusters) typically range from 0.3-0.4

  • Monitor time-dependent changes during reconstitution to assess rates

• Electron paramagnetic resonance (EPR):

  • [4Fe-4S]+ clusters (reduced state) exhibit characteristic EPR signals

  • The absence of signals in the oxidized state ([4Fe-4S]2+) is expected

  • Pattern distinctions between the reducing and auxiliary clusters provide insights into differential coordination environments

  • Quantify spin concentration to determine the proportion of clusters in active states

• Mössbauer spectroscopy:

  • Provides detailed information about the oxidation states of iron atoms

  • Can distinguish between different types of iron-sulfur clusters

  • Useful for confirming the presence of both [4Fe-4S] clusters in lipA

Based on studies with human LIAS, EPR analysis can reveal the likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center . This ordering information is crucial for optimizing reconstitution protocols and understanding the assembly pathway of functional lipA.

What approaches can resolve contradictory results between in vitro activity assays and in vivo functional studies of lipA?

Resolving contradictions between in vitro activity assays and in vivo functional studies of Salmonella paratyphi B lipA requires a systematic approach that bridges the gap between controlled laboratory conditions and complex biological environments:

• Identify sources of discrepancy:

  • Differences in redox environment between test tube and cellular conditions

  • Presence of additional cofactors or protein partners in vivo

  • Substrate accessibility and concentration variations

  • Post-translational modifications present only in cellular contexts

• Bridging experimental approaches:

  • Develop cell extract assays that maintain native protein interactions

  • Reconstruct minimal systems with known in vivo components

  • Use genetic complementation to validate biochemical findings

  • Employ in vivo activity reporters linked to lipoic acid production

• Reconciliation strategy:

Table 2: Systematic Approach for Resolving In Vitro vs. In Vivo Discrepancies

Studies of human LIAS have demonstrated that specific Fe-S cluster donor proteins are required for full enzymatic activity . Similar protein partners may be necessary for Salmonella paratyphi B lipA function in vivo, explaining potential discrepancies with simplified in vitro systems. By systematically addressing each potential source of variation, researchers can develop more physiologically relevant in vitro assays that better predict in vivo function.