Recombinant Staphylococcus aureus Methionine aminopeptidase (map)

What is Staphylococcus aureus Methionine aminopeptidase and why is it significant in antibacterial research?

Staphylococcus aureus Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for removing N-terminal methionine initiators from nascent proteins during protein synthesis. The enzyme plays an essential role in post-translational modifications necessary for protein translocation, activation, regulation, and degradation. Research has definitively established that MetAP activity is essential for bacterial cell viability, as knockout of the map gene in E. coli and pepM gene in S. typhimurium results in cellular death . This essentiality makes S. aureus MetAP an attractive antibacterial drug target, particularly given the significant disease burden of S. aureus infections in humans and animals . The development of specific inhibitors targeting S. aureus MetAP could potentially provide new antibacterial therapeutics against this important pathogen, which frequently develops resistance to conventional antibiotics.

How does the structure of S. aureus MetAP compare to human MetAPs, and what are the implications for selective inhibitor design?

Structural comparison between bacterial MetAPs and human MetAPs reveals important differences that can be exploited for developing selective inhibitors. While the catalytic domains of MetAPs are generally conserved across species, significant structural variations exist, particularly in the active site architecture. Human cells possess two types of MetAP enzymes (MetAP1 and MetAP2) with distinct structural features . Crystal structure analysis has shown that the active site of human MetAP1 is reduced in size compared to MetAP2, which explains why certain inhibitors like ovalicin target human MetAP2 but not MetAP1.

For S. aureus MetAP, the unique features of its active site and adjacent regions provide opportunities for selective targeting. These differences include variations in metal coordination sites, binding pocket dimensions, and surface topography. X-ray crystallography data of bacterial MetAPs (with over 65 protein crystal structures deposited in the PDB) have been instrumental in identifying these differences . When designing inhibitors, researchers should focus on the unique structural elements of S. aureus MetAP that differ from human homologs to minimize potential cross-reactivity and toxicity.

What expression systems yield optimal production of functional recombinant S. aureus MetAP?

The selection of an appropriate expression system is critical for obtaining high yields of soluble and active recombinant S. aureus MetAP. Based on established protocols for similar metalloenzymes, E. coli-based expression systems using pET vectors under T7 promoter control typically provide good yields. For optimal expression, several methodological considerations are important:

Expression Conditions Table:

When expressing recombinant MetAP, it's crucial to supplement the growth medium with appropriate metal ions, as MetAPs are metalloproteases that require metal cofactors for proper folding and catalytic activity. Co²⁺ has been identified as particularly effective for many bacterial MetAPs . Additionally, fusion tags such as His₆ facilitate purification while often enhancing solubility. For structural studies requiring high purity, TEV protease cleavage sites can be incorporated to remove the tag after initial purification steps.

What are the recommended methods for assaying recombinant S. aureus MetAP activity?

Several robust methods exist for measuring the enzymatic activity of recombinant S. aureus MetAP. The most widely used approaches include:

• Chromogenic/Fluorogenic Substrates: Methionyl analogs of 7-amino-4-methylcoumarin (AMC) and p-nitroaniline (pNA) provide continuous monitoring of amide bond cleavage . For example, Met-AMC releases fluorescent AMC upon cleavage, allowing sensitive real-time measurement of enzymatic activity.

• Peptide-Based Assays: Short synthetic peptides (3-5 amino acids) with N-terminal methionine serve as efficient substrates, with kcat/Km values of approximately 5×10⁵ M⁻¹ min⁻¹ . HPLC or mass spectrometry can be used to quantify the cleaved products.

• Coupled-Enzyme Assays: Novel coupled-enzyme assay methods link MetAP activity to a secondary reaction that produces a detectable signal, enhancing sensitivity for kinetic studies .

For accurate activity measurements, it's essential to control metal ion concentration, as excess metal ions can inhibit MetAP activity. Experiments have shown that in the presence of excess Co²⁺, a third Co²⁺ ion can bind in the active site region, explaining the inhibitory effect of excess metal cations . Optimal assay conditions typically include a buffer system at pH 7.5, physiological salt concentration, and carefully titrated metal cofactors.

How does the regulatory mechanism of methionine biosynthesis in S. aureus differ from other Bacillales?

S. aureus employs a unique and complex regulatory mechanism for methionine biosynthesis that differs significantly from other Bacillales species. While most Bacillales use S-box riboswitches to control methionine biosynthesis, S. aureus has evolved a rare combination of regulatory elements consisting of:

• T-box Riboswitch System: The S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch instead of the S-box riboswitch found in other Bacillales . This T-box riboswitch specifically interacts with uncharged initiator formylmethionyl-tRNA (tRNAᵢᶠᴹᵉᵗ) rather than elongator tRNAᴹᵉᵗ, suggesting a potential additional role in translation initiation control.

• CodY Repressor Control: Expression of the met leader RNA/metICFE-mdh operon is regulated by the repressor CodY, which binds upstream of the met leader RNA promoter . This represents an additional layer of transcriptional control not present in many other bacterial species.

• Stringent Response Integration: Methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis as part of the metabolic emergency circuit of the stringent response. This releases CodY from its binding site, thereby activating the met leader promoter .

• mRNA Decay Mechanisms: The full-length met leader/metICFE-mdh mRNA is subject to rapid degradation by nucleases involving RNase J2 when produced under methionine-limited conditions .

This multilayered regulation reflects the critical need for tight control of methionine metabolism in S. aureus, possibly due to its limited metabolic capacity to reuse methionine. Unlike Bacillus species, staphylococci lack both the methionine salvage and polyamine synthesis pathways, making methionine metabolism a potential metabolic vulnerability and an interesting target for anti-staphylococcal drug development .

What are the critical considerations for structural biology approaches to identify druggable pockets in S. aureus MetAP?

Advanced structural biology approaches have revealed important insights into S. aureus MetAP's conformational dynamics that can inform drug discovery efforts. When conducting structural studies of S. aureus MetAP, researchers should consider several key aspects:

• Cryptic Pocket Identification: Recent research employing Adaptive Bandit molecular dynamics simulations has successfully identified cryptic pockets in human MetAP-II that were not visible in static crystal structures . Similar approaches can be applied to S. aureus MetAP to identify hidden druggable sites that may only become accessible during protein dynamics. These cryptic pockets often represent excellent targets for allosteric inhibitors.

• Disordered Loop Regions: Particular attention should be paid to disordered loop regions that are often not resolved in crystal structures. In MetAP-II, analysis of conformational flexibility in such regions has revealed hidden cryptic pockets with druggable properties . Network analysis indicates that disordered loop regions can have direct signaling routes to the active site, suggesting potential for allosteric regulation.

• Metal Ion Considerations: The metal ion composition significantly affects the structural properties of the active site. Crystallization and structural studies should account for different physiologically relevant metal ions (Co²⁺, Mn²⁺, Fe²⁺) to capture the full spectrum of conformational states. A third metal binding site has been identified in the active site region of some MetAPs, explaining inhibitory effects of excess metal ions .

• Computational Methods: Molecular dynamics simulations, particularly enhanced sampling techniques, are valuable for exploring conformational landscapes beyond what static crystal structures reveal. These approaches can identify transient pockets and characterize their druggability, informing structure-based drug design strategies.

For researchers conducting these studies, it's recommended to combine multiple approaches, including X-ray crystallography, NMR for dynamic regions, computational modeling, and biophysical assays to validate predicted druggable sites.

How do kinetic parameters of S. aureus MetAP compare with MetAPs from other species, and what are the implications for substrate specificity?

Kinetic characterization of MetAPs reveals important differences in substrate preferences and catalytic efficiencies across species. While comprehensive kinetic data specifically for S. aureus MetAP is limited in the provided search results, comparative analysis with human and other bacterial MetAPs provides valuable insights.

Comparative Kinetic Parameters Table:

Studies on human MetAP2 have demonstrated that the most efficient substrates are peptides of three or more amino acids, with kcat/Km values of approximately 5×10⁵ M⁻¹ min⁻¹ . Site-directed mutagenesis studies found that MetAP enzymes generally recognize substrates containing one of seven amino acids with the smallest radii of gyration adjacent to the N-terminal methionine, while larger side chains prevent interaction with the enzyme .

When designing kinetic experiments for S. aureus MetAP, researchers should consider both methionyl-AMC/pNA substrates for initial screening and longer peptide substrates that more closely mimic natural substrates for detailed characterization. The nature of the P1' residue (the residue adjacent to methionine) significantly influences substrate efficiency and should be systematically varied to determine specificity profiles . This information is crucial for designing selective inhibitors that exploit differences in substrate binding pockets between bacterial and human MetAPs.

What experimental approaches can elucidate the role of S. aureus MetAP in bacterial virulence and host-pathogen interactions?

Understanding the connection between S. aureus MetAP activity and virulence requires sophisticated experimental approaches spanning molecular genetics, infection models, and systems biology. Several methodological approaches are particularly valuable:

• Conditional Knockout Systems: Since complete MetAP deletion is lethal, conditional expression systems using inducible promoters (e.g., controlled by isopropyl-β-thiogalactoside) can regulate MetAP levels . This allows researchers to study the effects of MetAP depletion on virulence factor expression and bacterial fitness in various environments.

• Experimental Evolution Models: Recent work has developed experimental evolution approaches for S. aureus adaptation to host environments. In one study, S. aureus strains representing major human epidemic clones were repeatedly passaged through macrophage cell lines, accumulating mutations and developing enhanced survival capabilities in macrophages and human blood . Similar approaches could be used to understand how MetAP function adapts under selective pressure.

• Animal Infection Models: Zebrafish infection models have proven useful for studying S. aureus pathogenesis and can reveal differences in virulence between wild-type and MetAP-modulated strains . These models allow for real-time visualization of infection dynamics and host-pathogen interactions.

• Proteomics Approaches: Since MetAP affects the N-terminal processing of numerous proteins, comparative proteomics between wild-type and MetAP-inhibited S. aureus can identify specific virulence factors and pathways most affected by MetAP activity. N-terminal proteomics techniques are particularly relevant for this application.

• Small Colony Variant (SCV) Analysis: S. aureus can develop small colony variants with altered phenotypes, including increased survival in macrophages and antibiotic resistance . Investigating whether MetAP activity influences SCV formation could provide insights into bacterial adaptation and persistence mechanisms.

Interestingly, experimental evolution studies have identified novel adaptive phenotypes in S. aureus that promote survival in macrophages and resistance to vancomycin . Similar approaches focused on MetAP function could reveal its role in bacterial adaptation to host defenses and antimicrobial treatments.

What challenges exist in developing selective inhibitors for S. aureus MetAP, and how might they be overcome?

Developing selective inhibitors for S. aureus MetAP presents several significant challenges that require sophisticated drug design approaches. The major challenges and potential solutions include:

• Selectivity vs. Human MetAPs: The primary challenge is achieving selectivity against bacterial MetAPs while avoiding inhibition of human MetAP1 and MetAP2. Structural analysis has revealed that human MetAP1 has a reduced active site size compared to MetAP2, explaining why some inhibitors like ovalicin target human MetAP2 but not MetAP1 . To overcome this, researchers should:

  • Focus on structural differences in both the active site and adjacent regions

  • Utilize structure-based design targeting S. aureus-specific binding pockets

  • Develop allosteric inhibitors targeting non-conserved regulatory sites

• Metal Cofactor Considerations: MetAPs are metalloenzymes, and their activity depends on metal cofactors. The type and number of metal ions affect both enzyme activity and inhibitor binding. In the presence of excess Co²⁺, a third Co²⁺ ion can bind in the active site region of some MetAPs, influencing enzyme activity . Inhibitor design should:

  • Consider the physiologically relevant metal cofactor in S. aureus

  • Account for changes in binding site geometry with different metals

  • Explore metal-chelating inhibitors that exploit differences in metal coordination

• Tackling Cryptic Pockets: Recent research using Adaptive Bandit molecular dynamics simulations has identified hidden cryptic pockets in MetAP-II that could be targeted by allosteric inhibitors . Similar approaches applied to S. aureus MetAP might:

  • Reveal unique druggable sites not apparent in static crystal structures

  • Identify allosteric sites with direct signaling routes to the active site

  • Enable the design of inhibitors that lock the enzyme in inactive conformations

• Leveraging Regulatory Mechanisms: S. aureus employs unique regulatory mechanisms for methionine metabolism involving CodY repressor, T-box riboswitches, and mRNA decay mechanisms . These regulatory elements could be exploited to:

  • Develop inhibitors that disrupt the interaction between the T-box riboswitch and tRNAᵢᶠᴹᵉᵗ

  • Target the stringent response pathway that controls MetAP expression

  • Design compounds that interfere with both MetAP activity and its regulation

By combining structural biology, dynamics simulations, and deep understanding of S. aureus-specific regulatory mechanisms, researchers can develop more selective and effective inhibitors targeting this essential enzyme.

How do metal cofactors affect the activity, stability, and inhibition profile of recombinant S. aureus MetAP?

Metal cofactors play a crucial role in determining the activity, stability, and inhibition profile of MetAP enzymes. For recombinant S. aureus MetAP, several key considerations regarding metal cofactors include:

Metal Effects on MetAP Activity Table:

When working with recombinant S. aureus MetAP, researchers should carefully consider the metal cofactor used in their experiments, as it will significantly impact experimental outcomes and the translation of in vitro results to in vivo applications.

How might the unique T-box riboswitch system in S. aureus methionine biosynthesis be exploited as an antibacterial target?

The unusual regulatory mechanism of methionine biosynthesis in S. aureus presents a promising but underexplored target for novel antibacterial strategies. Unlike other Bacillales that employ S-box riboswitches, S. aureus utilizes a T-box riboswitch system that specifically interacts with uncharged initiator formylmethionyl-tRNA (tRNAᵢᶠᴹᵉᵗ) . This unique regulatory feature offers several potential avenues for therapeutic intervention:

• T-box Riboswitch Targeting: Small molecules that mimic the structure of uncharged tRNAᵢᶠᴹᵉᵗ could potentially bind to the T-box riboswitch, disrupting its normal regulatory function. This could lead to dysregulation of methionine biosynthesis and bacterial growth inhibition. The specificity of the S. aureus T-box riboswitch for tRNAᵢᶠᴹᵉᵗ (with weak binding to elongator tRNAᴹᵉᵗ) provides a basis for selective targeting .

• CodY Repressor Modulation: The methionine biosynthesis pathway in S. aureus is under the control of the repressor CodY, which binds upstream of the met leader RNA promoter . Compounds that enhance CodY binding or prevent its release during methionine depletion could potentially suppress methionine biosynthesis, thereby inhibiting bacterial growth.

• Exploiting Stringent Response Connection: Methionine depletion in S. aureus activates RelA-dependent (p)ppGpp alarmone synthesis as part of the stringent response . Targeting the interaction between methionine metabolism and the stringent response could provide a novel approach to disrupt bacterial adaptation to nutrient limitation.

• mRNA Decay Mechanism Interference: The full-length met leader/metICFE-mdh mRNA in S. aureus is subject to rapid degradation by nucleases involving RNase J2 . Modulating this decay mechanism could potentially disrupt the tight regulation of methionine biosynthesis, leading to metabolic imbalances.

Research in this area would benefit from high-throughput screening approaches to identify compounds that interact with the T-box riboswitch or affect CodY-mediated regulation. Structural studies of the T-box riboswitch-tRNAᵢᶠᴹᵉᵗ complex would further facilitate rational drug design targeting this unique regulatory system.

What is the relationship between S. aureus MetAP function and small colony variant (SCV) formation in clinical infections?

Small colony variants (SCVs) of S. aureus represent a significant clinical challenge due to their association with persistent infections and antibiotic resistance. Recent research has identified novel adaptive phenotypes in S. aureus, including a previously undescribed SCV phenotype characterized by hyper-pigmentation . Understanding the potential relationship between MetAP function and SCV formation could provide valuable insights for clinical management of S. aureus infections:

• MetAP's Role in Proteome Remodeling During Adaptation: As MetAP processes newly synthesized proteins, alterations in its activity during adaptation to stressful environments (such as within macrophages or in the presence of antibiotics) could contribute to the proteome remodeling associated with SCV formation. Experimental evolution studies have shown that S. aureus strains passaged through macrophage cell lines accumulate mutations in various genomic loci, leading to enhanced survival in macrophages and human blood, along with antibiotic resistance .

• Potential Connection to Stress Response Pathways: The recently described SCV phenotype resulting from a missense mutation in rsbW suggests involvement of the stress response sigma factor SigB . Given that methionine metabolism in S. aureus is integrated with the stringent response pathway , there may be interconnections between MetAP function, stress responses, and SCV formation that warrant investigation.

• Implications for Persistent Infections: SCVs are known to exhibit heightened intracellular persistence, which contributes to chronic infections. The conditional nature of the novel SCV phenotype, which rapidly converts to a large colony variant in nutrient-replete conditions through spontaneous sigB deletion events , suggests complex regulatory mechanisms that might involve methionine metabolism and MetAP function.

• Research Approaches: To investigate these connections, researchers could employ:

  • Comparative proteomics between wild-type and SCV S. aureus strains, focusing on N-terminal processing differences

  • Experimental evolution studies with MetAP inhibitors to determine if altered MetAP function influences SCV formation

  • Analysis of MetAP expression and activity in clinical SCV isolates versus normal phenotype strains

  • Investigation of methionine metabolism in the context of host-pathogen interactions, particularly within macrophages

The identification of similar sigB deletion events in clinical S. aureus isolates suggests that the regulatory mechanisms underlying SCV formation and reversion occur during clinical infections, making this an important area for further investigation.