Recombinant Salmonella schwarzengrund Peptide deformylase (def)

What is the function of peptide deformylase in Salmonella species?

Peptide deformylase (PDF) in Salmonella species, including S. schwarzengrund, functions as an essential metalloprotease responsible for removing the N-formyl group from nascent polypeptide chains during protein synthesis. This post-translational modification is critical because bacterial protein synthesis begins with N-formylmethionine, and the formyl group must be removed for proper protein maturation and function. Studies with S. enterica serovar Typhimurium demonstrate that PDF is essential for bacterial viability, as it catalyzes a required step in protein maturation . The enzyme contains a metal center (typically iron) that is crucial for catalytic activity, and its function is susceptible to inhibition by various agents including nitric oxide and other oxidizing agents, representing a mechanism by which host defenses can target bacterial growth .

How is recombinant Salmonella PDF typically expressed and purified?

Expression of recombinant Salmonella PDF typically involves cloning the def gene into an expression vector such as pET28c with appropriate affinity tags (commonly hexahistidine). For S. Typhimurium PDF, researchers have successfully amplified the 510-bp def gene using sequence-specific primers incorporating restriction sites for subsequent cloning . The recombinant construct is then transformed into E. coli BL21(DE3) for protein expression .

For purification, cells are typically lysed by sonication in buffer containing protease inhibitors (such as PMSF, pepstatin, and leupeptin) . The lysate is clarified by centrifugation, and the supernatant is subjected to nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Purification protocols for Salmonella PDF often involve washing with 50 mM HEPES (pH 7.0) containing 100 mM NaCl and 20 mM imidazole, followed by elution with the same buffer containing 200 mM imidazole . To preserve enzyme activity, imidazole can be removed by dialysis against 50 mM HEPES (pH 7.0) with 100 mM NaCl .

What methods are used to assess the enzymatic activity of recombinant PDF?

PDF enzyme activity is commonly assessed through a spectrophotometric assay using synthetic substrates such as N-formyl-Met-Ala. In a standard protocol, the enzyme (typically 50-125 ng) is incubated with substrate (5 mM for single-point assays or 0-20 mM for kinetic studies) in 50 mM HEPES buffer (pH 7.0) containing 100 mM NaCl at 30°C for 30 minutes . The reaction is terminated by adding 4% HClO₄, and the reaction mixture is further incubated with trinitrobenzene sulfonic acid (TNBSA) reagent at 37°C for 2 hours .

The TNBSA reagent reacts with the primary amine generated by deformylation, forming a highly chromogenic derivative that can be quantified by measuring absorbance at 340 nm using an ELISA reader . Enzyme activity is typically calculated as nanomoles of free amino group produced per minute per milligram of protein, determined using standard curves prepared with L-methionine . This assay enables researchers to determine both the specific activity of PDF preparations and kinetic parameters when performed with varying substrate concentrations.

What are the key structural features of Salmonella PDF enzymes?

Salmonella PDF enzymes share several key structural features with other bacterial PDFs. The enzyme contains a crucial metal-binding site with a characteristic motif involving two histidines and one cysteine (Cys-90 in S. Typhimurium PDF) . This metal-coordinating cysteine is particularly significant as it represents a target for inhibition by various agents, including nitric oxide through S-nitrosylation .

In addition to the catalytic metal-binding site, Salmonella PDFs contain non-catalytic residues that influence enzyme behavior. For example, Cys-130 in S. Typhimurium PDF, while not directly involved in catalysis, significantly impacts the enzyme's susceptibility to oxidative stress . Comparative studies between S. Typhimurium PDF and M. tuberculosis PDF have demonstrated that this non-catalytic cysteine, which corresponds to Met-145 in the M. tuberculosis enzyme, is responsible for the markedly different sensitivities of these enzymes to oxidizing agents like H₂O₂ .

How does nitric oxide (NO·) affect the activity of Salmonella PDF?

Nitric oxide (NO·) inhibits Salmonella peptide deformylase through two distinct mechanisms, representing an important aspect of host-pathogen interactions. First, NO· directly inhibits PDF by S-nitrosylating the metal-binding Cys-90 residue, as demonstrated in S. Typhimurium . This modification was confirmed through biotin switch assays, where S-nitrosyl groups are exchanged with biotin and subsequently detected by immunoblotting . When the Cys-90 residue was mutated to serine (PDF-C90S), no significant biotinylation was observed after NO· treatment, confirming this residue as the primary target for S-nitrosylation .

Second, NO· indirectly inhibits PDF by mobilizing zinc from metalloproteins, increasing free intracellular zinc that subsequently causes mismetallation of the iron-containing PDF . This zinc-dependent inhibition was demonstrated in S. Typhimurium strains lacking zinc exporters (ΔzntA ΔzitB), which showed enhanced sensitivity to NO· . Importantly, overexpression of PDF restored growth of the ΔzntA ΔzitB strain relative to wild-type in the presence of NO·, confirming that zinc-dependent PDF inhibition contributes significantly to the enhanced NO· sensitivity of zinc export-deficient strains .

These findings reveal PDF as a critical bacterial protein subjected to both direct and indirect inactivation by host-derived NO·, providing insight into the mechanisms of nitric oxide's antimicrobial activity .

What role do non-catalytic cysteine residues play in the oxidative sensitivity of Salmonella PDF?

Non-catalytic cysteine residues play a crucial role in determining the oxidative sensitivity of Salmonella PDF, despite being located away from the active site. Comparative studies between S. Typhimurium PDF (sPDF) and M. tuberculosis PDF (mPDF) revealed striking differences in their susceptibility to oxidizing agents like H₂O₂, with IC₅₀ values ranging from nanomolar levels for sPDF to millimolar levels for mPDF .

Sequence analysis identified that Cys-130 in sPDF corresponds to Met-145 in mPDF, both positioned distant from the active site . Through site-directed mutagenesis, researchers demonstrated that swapping methionine with cysteine in mPDF (creating the M145C variant) drastically decreased the IC₅₀ for H₂O₂ and increased the metal dissociation rate compared to wild-type mPDF . Matrix-assisted laser desorption ionization (MALDI) analysis of trypsin-digested fragments containing the mutated residue confirmed increased susceptibility to oxidation .

Conversely, creating a double mutant of sPDF (C130M-V63C) to incorporate residues identical to those in mPDF increased the IC₅₀ for H₂O₂ compared to wild-type sPDF . Importantly, the oxidation state of cysteines in this double mutant remained unaffected during H₂O₂ treatment . These findings establish the critical role of non-catalytic cysteine/methionine residues in determining enzymatic sensitivity to oxidative stress, explaining the behavioral differences between bacterial PDFs under oxidative conditions.

How can researchers differentiate between direct inhibition and metal-dependent inhibition mechanisms of Salmonella PDF?

Differentiating between direct inhibition and metal-dependent inhibition mechanisms of Salmonella PDF requires a systematic experimental approach. For direct inhibition assessment, researchers can measure PDF activity in purified protein extracts after treatment with inhibitors such as nitric oxide donors . Since inhibitors are added ex vivo after extract purification, any observed inhibition results from direct enzyme modification rather than cellular processes like metal mismetallation .

To identify specific mechanisms of direct inhibition, site-directed mutagenesis of potential target residues is invaluable. For example, mutation of the metal-binding Cys-90 to serine (PDF-C90S) in S. Typhimurium PDF abolished S-nitrosylation by NO·, confirming this residue as the primary target for direct inhibition . Similar approaches can be used to evaluate other potential inhibition mechanisms.

For metal-dependent inhibition, researchers should examine PDF activity in bacterial strains with altered metal homeostasis. Studies with S. Typhimurium demonstrated that strains lacking zinc exporters (ΔzntA ΔzitB) showed enhanced PDF inhibition during zinc overload or nitrosative stress due to mismetallation . Complementation experiments, where PDF is overexpressed in these strains, can confirm the role of mismetallation in enzyme inhibition .

Interestingly, researchers have observed that zinc-substituted PDF obtained from zinc-treated ΔzntA ΔzitB strains was not inhibited by NO·, suggesting that metal identity influences susceptibility to direct inhibition . This observation highlights the complex interplay between direct modification and metal-dependent mechanisms.

What are the optimal conditions for expressing and purifying active recombinant Salmonella PDF?

The optimal conditions for expressing and purifying active recombinant Salmonella PDF require careful consideration of several factors to maintain enzyme integrity and activity. Based on protocols developed for S. Typhimurium PDF, the following approach is recommended:

For maintaining iron-containing enzyme activity, it's crucial to include reducing agents in buffers and consider working under anaerobic conditions to prevent oxidation of the metal center. Purified enzyme should be stored in small aliquots at -80°C to minimize freeze-thaw cycles that can lead to activity loss.

What considerations are important when designing site-directed mutagenesis experiments for Salmonella PDF?

When designing site-directed mutagenesis experiments for Salmonella PDF, researchers should prioritize several key considerations to ensure meaningful results. Based on successful studies with S. Typhimurium PDF and M. tuberculosis PDF, the following approach is recommended:

First, identify target residues based on functional importance or comparative analysis. Critical residues include the metal-coordinating Cys-90 and non-catalytic Cys-130 in S. Typhimurium PDF, which influence enzyme activity and oxidative sensitivity, respectively . Researchers have successfully employed PCR-based overlap extension methods using external primers (e.g., CK1 and CK2 for sPDF) and internal primers incorporating desired mutations .

The selection of amino acid substitutions is crucial. For metal-binding residues like Cys-90, serine substitution (C90S) maintains similar structure while eliminating the thiol group, enabling investigation of S-nitrosylation mechanisms . For studying oxidative sensitivity, substitutions that change chemical properties (e.g., replacing Cys-130 with Met to mimic M. tuberculosis PDF) have proven valuable .

Researchers should confirm mutations by DNA sequencing and validate protein expression using SDS-PAGE and western blotting. Comprehensive characterization of mutant proteins should include assessment of metal content, enzymatic activity, and susceptibility to inhibitors to establish structure-function relationships .

How should researchers approach studying PDF in the context of host-pathogen interactions?

Studying PDF in the context of host-pathogen interactions requires approaches that bridge biochemical characterization with infection biology. Researchers investigating Salmonella PDF should consider the following strategies:

First, understand host defense mechanisms targeting PDF. Studies with S. Typhimurium have demonstrated that host-derived nitric oxide (NO·) inhibits PDF through dual mechanisms: direct S-nitrosylation of Cys-90 and indirect zinc mismetallation . These findings highlight the importance of examining PDF within the context of host immune responses.

To investigate PDF inhibition during infection, researchers can use macrophage infection models that generate nitrosative stress. Comparing the growth of wild-type Salmonella with strains overexpressing PDF or PDF mutants resistant to specific inhibition mechanisms can reveal the contribution of PDF inhibition to host defense . For example, PDF overexpression partially restored growth of S. Typhimurium zinc export mutants (ΔzntA ΔzitB) under nitrosative stress, confirming the role of PDF inhibition in NO·-mediated growth suppression .

Researchers should also consider the differential effects of various host-derived antimicrobial mechanisms. While PDF overexpression rescued growth during zinc overload, it failed to restore normal growth rates under nitrosative stress and actually exacerbated growth inhibition relative to controls . This suggests complex interactions between different inhibition mechanisms that warrant further investigation.

What are common issues encountered when working with iron-containing PDF enzymes?

Working with iron-containing PDF enzymes presents several challenges that researchers must address to obtain reliable results. One primary concern is oxidative inactivation of the iron center, which occurs readily under aerobic conditions. The Fe(II) form required for catalytic activity can oxidize to Fe(III), rendering the enzyme inactive . This sensitivity to oxidation is particularly pronounced in Salmonella PDF compared to more resistant enzymes like M. tuberculosis PDF .

Another significant challenge is metal substitution or mismetallation. Under certain conditions, particularly during nitrosative stress or disrupted zinc homeostasis, zinc can replace iron in the PDF active site . While the enzyme can accommodate different metals, this substitution significantly reduces catalytic activity. In S. Typhimurium lacking zinc exporters (ΔzntA ΔzitB), researchers observed zinc mismetallation of PDF when exposed to excess zinc or nitric oxide, highlighting the challenges in maintaining the native metal content .

Furthermore, different PDF variants show variable susceptibility to inhibition by oxidizing agents. S. Typhimurium PDF is inhibited by H₂O₂ at nanomolar concentrations, while M. tuberculosis PDF requires millimolar concentrations for comparable inhibition . These differences stem from non-catalytic residues like Cys-130 in S. Typhimurium PDF, which influence oxidative sensitivity despite being distant from the active site .

How can researchers address inconsistent results when measuring PDF enzyme activity?

Inconsistent results when measuring PDF enzyme activity can stem from multiple sources that researchers must systematically address. A primary concern is the variable metal content of enzyme preparations. Since PDF activity depends on the metal cofactor (typically iron), variations in metallation state can cause significant activity differences . Researchers should implement consistent methods for metal reconstitution and verify metal content using appropriate analytical techniques.

The oxidation state of the enzyme also critically affects activity measurements. Iron in PDF is active in the Fe(II) state but can readily oxidize to Fe(III) during purification and storage . Implementing anaerobic techniques, including buffer degassing and the inclusion of reducing agents, helps maintain the active state. For consistent results, all activity assays should be performed under identical redox conditions.

Post-translational modifications like S-nitrosylation can also affect PDF activity . Even trace amounts of nitrosylating agents can modify the critical Cys-90 residue, reducing enzyme activity. Researchers should carefully control experimental conditions to avoid inadvertent enzyme modification and consider including controls to detect such modifications.

Additionally, the spectrophotometric assay commonly used for PDF activity measurement can be subject to interference. The TNBSA reagent reacts with primary amines, so contamination with free amines in buffers or enzyme preparations can produce false-positive results . Careful buffer preparation and appropriate blank controls are essential for accurate activity determination.

How might studies of PDF inhibition inform the development of novel antimicrobial strategies?

Studies of PDF inhibition offer promising avenues for developing novel antimicrobial strategies against Salmonella and other bacterial pathogens. The dual mechanisms of PDF inhibition by nitric oxide—direct S-nitrosylation of Cys-90 and indirect zinc mismetallation—provide a framework for designing inhibitors that target this essential enzyme . Compounds that can either modify the metal-binding cysteine or promote mismetallation could effectively suppress bacterial growth by inhibiting protein maturation.

The discovery that non-catalytic residues like Cys-130 in S. Typhimurium PDF significantly influence oxidative sensitivity offers additional targeting opportunities . Inhibitors could be designed to exploit these residues, potentially triggering oxidative inactivation of the enzyme. The striking differences in H₂O₂ sensitivity between S. Typhimurium PDF (nanomolar IC₅₀) and M. tuberculosis PDF (millimolar IC₅₀) highlight how species-specific variations can be leveraged for selective targeting .

Furthermore, the observation that PDF overexpression can rescue growth during zinc overload but exacerbates growth inhibition under nitrosative stress suggests complex interactions that could be exploited through combination therapies . Agents that simultaneously disrupt metal homeostasis and promote nitrosative stress might achieve synergistic inhibition of bacterial growth by targeting PDF through multiple mechanisms.

Future research should focus on developing PDF inhibitors that can penetrate bacterial cells, resist efflux, and maintain stability in the infection environment. Structure-based drug design informed by the detailed understanding of PDF inhibition mechanisms will be instrumental in advancing these novel antimicrobial strategies.

What are the implications of metal substitution in PDF for bacterial survival during host infection?

Metal substitution in PDF has significant implications for bacterial survival during host infection, representing both a vulnerability and a potential adaptation mechanism. During infection, host cells generate nitric oxide, which mobilizes zinc from bacterial metalloproteins, increasing free intracellular zinc that can mismetallate iron-containing enzymes like PDF . This mismetallation reduces PDF activity, contributing to growth inhibition of pathogens like Salmonella.

The differential sensitivity of bacterial species to this process may influence their pathogenicity and persistence in host environments. Bacteria with more robust mechanisms for maintaining proper PDF metallation, or with PDFs less susceptible to functional impairment upon metal substitution, might exhibit enhanced survival during infection. Conversely, pathogens more vulnerable to PDF mismetallation could be more effectively controlled by host defenses.

Interestingly, zinc-substituted PDF obtained from zinc-treated ΔzntA ΔzitB S. Typhimurium was not inhibited by nitric oxide, unlike the iron-containing enzyme . This suggests that while metal substitution reduces enzymatic activity, it might also confer resistance to certain inhibition mechanisms. This complex interplay could influence the evolution of bacterial responses to host defenses.

Future research should investigate whether different Salmonella serovars, including S. schwarzengrund, exhibit variations in PDF metal preference, susceptibility to mismetallation, and functional consequences of metal substitution. Such studies could reveal adaptations that contribute to virulence and persistence during infection, potentially identifying new targets for therapeutic intervention.