Recombinant Synechocystis sp. UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase (lpxC)

What is the physiological role of LpxC in Synechocystis sp.?

LpxC catalyzes the deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine in the lipid A biosynthetic pathway. This reaction represents the committed step in lipopolysaccharide (LPS) biosynthesis, making it essential for bacterial survival. In Synechocystis sp., as in other Gram-negative bacteria, LpxC plays a crucial role in cell envelope biogenesis and maintaining membrane integrity. The enzyme's function is particularly critical because it sits at a key regulatory point between LPS production and phospholipid biosynthesis, effectively controlling the balance between these two essential membrane components. This balance is vital for proper membrane architecture and function, with disruptions potentially leading to membrane instability and cell death .

Notably, LpxC has no sequence homology to known mammalian deacetylases or amidases, making it an excellent target for antibiotic development against Gram-negative bacterial infections. The enzyme's essentiality coupled with its absence in human cells creates an opportunity for selective toxicity - a fundamental principle in antibiotic design.

How does LpxC from Synechocystis sp. compare with LpxC enzymes from other organisms?

While the specific characteristics of Synechocystis sp. LpxC have not been fully elucidated, we can draw comparisons based on conserved features of LpxC enzymes across different bacterial species. LpxC is highly conserved among Gram-negative bacteria at the functional level, though sequence divergence exists. The enzyme typically features a novel α/β fold, a unique zinc-binding motif, and a hydrophobic passage that accommodates the acyl chain of the substrate .

A distinctive feature of LpxC is its dual metal ion dependency. The enzyme can function with either Zn(II) or Fe(II) as cofactors, with Fe(II)-LpxC demonstrating 3-5 fold higher catalytic efficiency than Zn(II)-LpxC . This metal preference may vary between species and could represent an adaptation to different environmental conditions or metabolic requirements.

In transcriptional regulation, Synechocystis sp. PCC 6803 contains numerous transcriptional regulators with diverse functions, as evidenced by metabolomic profiling of knockout mutants . While specific regulators of lpxC in Synechocystis haven't been explicitly identified in the search results, the regulation likely involves both transcriptional control and post-translational mechanisms similar to those in other bacteria.

What is known about post-translational regulation of LpxC in bacteria?

Post-translational regulation of LpxC occurs primarily through proteolytic degradation mediated by the FtsH protease. This regulation is critical for maintaining the proper balance between LPS and phospholipid biosynthesis. When LpxC activity is inhibited by specific compounds, the enzyme becomes stabilized, suggesting that binding of inhibitors prevents FtsH-mediated degradation .

In experiments with E. coli, treatment with LpxC inhibitors resulted in a strong increase in LpxC abundance without corresponding increases in LPS levels, indicating that the inhibitors inactivate the enzyme while protecting it from degradation . The half-life of LpxC was significantly extended in the presence of inhibitors, with protein levels remaining stable for at least two hours compared to rapid degradation in control conditions.

This regulatory mechanism represents a sophisticated control system that allows bacteria to fine-tune their membrane composition in response to environmental conditions. Understanding these regulatory mechanisms in Synechocystis sp. specifically would require targeted experiments examining LpxC stability and turnover under various conditions.

What are the optimal expression systems for recombinant Synechocystis sp. LpxC?

For successful expression of recombinant Synechocystis sp. LpxC, several expression systems can be considered, with E. coli being the most commonly used for bacterial metalloenzymes. Based on methodological approaches for similar enzymes, the following strategies are recommended:

Table 1: Expression Systems for Recombinant LpxC Production

When expressing LpxC, several critical factors should be considered:

• Metal supplementation: Include ZnSO₄ (10-50 μM) or FeSO₄ (10-50 μM) in the growth medium to ensure proper metal incorporation, as LpxC requires either Zn(II) or Fe(II) for activity .

• Temperature optimization: Lower induction temperatures (16-20°C) generally improve proper folding and solubility of metalloenzymes.

• Affinity tags: An N-terminal His₆-tag is commonly used, though its placement should be carefully considered to avoid interference with metal binding or catalytic activity.

• Codon optimization: Consider codon optimization of the Synechocystis sp. gene for expression in the chosen host organism.

What purification strategy yields the highest activity for recombinant LpxC?

A multi-step purification approach is recommended to obtain highly active recombinant LpxC:

Table 2: Purification Protocol for Recombinant LpxC

Throughout purification, it's essential to avoid metal chelators like EDTA that could strip the essential metal cofactor from LpxC. Additionally, including a low concentration (5-10 μM) of the appropriate metal ion (Zn²⁺ or Fe²⁺) in the purification buffers can help maintain metalloenzyme activity .

Activity assays should be performed at each purification step to track enzyme activity and ensure that the final preparation retains full catalytic function. If activity loss occurs during purification, metal reconstitution might be necessary.

How can I verify proper metal incorporation and optimize the metal cofactor for maximal activity?

Verifying proper metal incorporation is crucial for ensuring maximal LpxC activity. Based on studies showing that LpxC can function with either Zn(II) or Fe(II), with Fe(II)-LpxC demonstrating 3-5 fold higher catalytic efficiency , both metal forms should be evaluated:

Analytical Methods for Metal Content Determination:

• Inductively Coupled Plasma Mass Spectrometry (ICP-MS) provides quantitative analysis of metal content with high sensitivity. This method can determine the metal-to-protein ratio, ideally approaching 1:1 for LpxC.

• Colorimetric Assays specific for zinc, as mentioned in the literature, can provide a simpler approach for routine analysis .

• Activity Assays with Metal Chelators: Comparing enzyme activity before and after treatment with metal chelators like dipicolinic acid (DPA) can confirm metal dependency. Complete inhibition by chelators followed by restoration of activity with specific metal ions confirms the metal requirement .

Metal Reconstitution Protocol:

• Remove bound metal by dialyzing the protein against buffer containing 5-10 mM dipicolinic acid at 4°C.

• Remove the chelator by dialysis against metal-free buffer.

• Add 2-5 molar equivalents of ZnSO₄ or FeSO₄ to the protein.

• Remove excess metal by dialysis or gel filtration.

• Quantify metal content and measure enzyme activity.

Comparing Zn(II) vs. Fe(II) Forms:

Given that Fe(II)-LpxC shows higher catalytic efficiency , it's worth preparing both metal forms and comparing their kinetic parameters. For Fe(II)-LpxC, special care must be taken to prevent oxidation by working under anaerobic conditions or including reducing agents in the buffers.

What are the optimal assay conditions for measuring Synechocystis sp. LpxC activity?

Optimal assay conditions for LpxC activity measurement should be established through systematic optimization. Based on general characteristics of LpxC enzymes, the following conditions are recommended as starting points:

Table 3: Recommended Assay Conditions for LpxC Activity

The physiological substrate for LpxC, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine, is strongly preferred over the simple substrate UDP-GlcNAc, with a 5×10⁶-fold difference in catalytic efficiency (kcat/KM) . Therefore, using the physiological substrate is strongly recommended for accurate activity measurements, despite the synthetic challenges.

What are the kinetic parameters of LpxC and how are they affected by metal cofactor choice?

While specific kinetic parameters for Synechocystis sp. LpxC have not been reported in the search results, general trends for LpxC enzymes provide valuable insights. The most significant factor affecting kinetic parameters is the choice of metal cofactor:

Table 4: Impact of Metal Cofactor on LpxC Kinetic Parameters

The higher catalytic efficiency of Fe(II)-LpxC is attributed to the higher coordination number of Fe(II) compared to Zn(II), as determined by X-ray absorption spectroscopy (XAS) . This structural difference likely affects substrate positioning and transition state stabilization in the active site.

To determine these parameters for Synechocystis sp. LpxC specifically, researchers should conduct steady-state kinetic analyses with varying substrate concentrations under optimal assay conditions, comparing both Zn(II) and Fe(II) forms of the enzyme.

What methods are available for high-throughput screening of LpxC inhibitors?

Several assay methods can be adapted for high-throughput screening of LpxC inhibitors, each with specific advantages and limitations:

Table 5: Assay Methods for High-Throughput Screening of LpxC Inhibitors

For primary screening, fluorescence-based or thermal shift assays are most suitable due to their high throughput and relatively simple setup. QSAR modeling approaches, as described in one study that built 24 classification models using PubChem and MACCS fingerprints with various machine learning algorithms, can complement experimental screening by predicting inhibitory activity .

Secondary assays should include orthogonal methods to confirm hits and eliminate false positives. The identification of "consensus activity cliff generators" - compounds where small structural changes lead to large differences in activity - can be particularly valuable for structure-activity relationship studies .

What structural features define LpxC and how do they contribute to catalysis?

LpxC possesses several unique structural features that distinguish it from other metalloenzymes and are crucial for its catalytic function:

• Novel α/β fold: The structure of LpxC reveals a previously undescribed protein fold that provides the framework for its specialized function .

• Unique zinc-binding motif: Unlike conventional zinc metalloenzymes, LpxC utilizes a distinctive metal-binding motif to coordinate the catalytic zinc ion .

• Hydrophobic passage: A defining feature of LpxC is a hydrophobic passage that accommodates the acyl chain of the substrate or inhibitors . This passage is critical for substrate recognition and binding, as evidenced by the 5×10⁶-fold preference for the acylated substrate over the deacylated form .

• Flexible active site motifs: Mobility and dynamics in structural elements near the active site play key roles in substrate capture . This conformational flexibility likely facilitates proper positioning of the substrate for catalysis.

The catalytic mechanism of LpxC involves the zinc ion activating a water molecule for nucleophilic attack on the acetyl group of the substrate. The metal ion also stabilizes the tetrahedral oxyanion intermediate formed during the reaction. Specific active site residues, though not explicitly detailed in the search results, would be involved in substrate binding, transition state stabilization, and proton transfer steps.

The dual metal specificity of LpxC adds another layer of complexity to its mechanism. The higher coordination number of Fe(II) compared to Zn(II) likely results in subtle but significant differences in the active site geometry, explaining the higher catalytic efficiency of Fe(II)-LpxC .

How does inhibitor binding affect LpxC structure and stability?

Inhibitor binding to LpxC induces significant changes in both the structure and stability of the enzyme, with implications for its cellular regulation:

• Protection from proteolysis: Treatment of E. coli with LpxC inhibitors results in elevated LpxC levels due to protection from FtsH-mediated degradation . All five tested inhibitors (CHIR-090, L-161,240, PF-05081090, PF-04753299, and BB-78485) stabilized LpxC, with the signal remaining stable for at least two hours after inhibitor addition .

• Conformational changes: While specific details aren't provided in the search results, the binding of substrate-analog inhibitor TU-514 to LpxC from Aquifex aeolicus was studied by solution NMR, suggesting that inhibitor binding induces conformational changes in the enzyme .

• Impact on metal coordination: Hydroxamate-containing inhibitors likely interact directly with the metal center, as their inhibition suggested the presence of a metal ion cofactor . This interaction potentially alters the metal coordination geometry.

• Differential cellular responses: Despite targeting the same enzyme, different LpxC inhibitors elicit distinct cellular responses beyond their direct effect on LpxC . This suggests that subtle differences in how inhibitors bind and affect LpxC structure can propagate to broader cellular effects.

Understanding these structural changes is crucial for rational inhibitor design. The solution structure of LpxC in complex with TU-514 provides a foundation for structure-based drug design efforts targeting this enzyme .

What is known about the catalytic mechanism of LpxC?

Based on biochemical and structural studies, a catalytic mechanism for LpxC has been proposed , though specific details are not fully elaborated in the search results. The general features of the mechanism likely include:

• Metal-activated water: The zinc or iron ion coordinates a water molecule, lowering its pKa and generating a hydroxide nucleophile.

• Nucleophilic attack: This activated water/hydroxide attacks the carbonyl carbon of the N-acetyl group of the substrate.

• Tetrahedral intermediate: A tetrahedral oxyanion intermediate forms, stabilized by the metal ion and potentially by hydrogen bonding to active site residues.

• Proton transfer: Proton transfer steps, facilitated by active site residues, lead to breakdown of the tetrahedral intermediate.

• Product release: The deacetylated product is released, completing the catalytic cycle.

The significantly higher preference for the physiological substrate containing the R-3-hydroxymyristoyl chain (5×10⁶-fold higher kcat/KM compared to UDP-GlcNAc) indicates that proper positioning of the substrate through hydrophobic interactions with the acyl chain is critical for efficient catalysis.

The higher catalytic efficiency of Fe(II)-LpxC compared to Zn(II)-LpxC (3-5 fold difference) suggests that the precise geometry of the metal center and its coordination sphere plays a significant role in optimizing the catalytic mechanism.

How do different classes of LpxC inhibitors compare in their potency and mechanism?

Various LpxC inhibitors have been developed and characterized, with notable differences in potency, mechanism, and cellular effects:

Table 6: Comparison of LpxC Inhibitors

Despite targeting the same enzyme, these inhibitors elicit both common and distinct cellular responses. All five tested compounds changed cell shape and stabilized LpxC, suggesting impairment of FtsH-mediated turnover . Four of the five compounds led to accumulation of lyso-PE, a cleavage product of phosphatidylethanolamine, indicating an imbalance between LPS and phospholipid biosynthesis .

Interestingly, compound-specific marker proteins belonged to different functional categories, including stress responses, nucleotide or amino acid metabolism, and quorum sensing . This suggests that subtle differences in how inhibitors interact with LpxC can lead to distinct downstream effects.

What structure-activity relationships have been identified for LpxC inhibitors?

Structure-activity relationship (SAR) studies for LpxC inhibitors have been approached through both experimental and computational methods. One study applied quantitative structure-activity relationship (QSAR) modeling to predict inhibitory activity of LpxC inhibitors .

Key findings from QSAR modeling include:

• Model performance: The best model using PubChem fingerprint was the Extremely Gradient Boost model (accuracy on the training set: 0.937; accuracy on the 10-fold cross-validation set: 0.795; accuracy on the test set: 0.799) .

• Alternative fingerprinting: Using MACCS fingerprint, the Random Forest model performed best (accuracy on the training set: 0.955; accuracy on the 10-fold cross-validation set: 0.803; accuracy on the test set: 0.785) .

• Activity cliffs: Eight "consensus activity cliff generators" were identified that are highly informative for SAR investigations . These represent compounds where small structural changes lead to large differences in activity.

While specific structural features enhancing inhibitory activity aren't detailed in the search results, the application of various machine learning algorithms suggests complex structure-activity relationships that can be modeled computationally.

The observed differences in potency among the five inhibitors tested in one study (with MICs ranging from 0.2 to 5 μg/ml) further indicate that structural variations significantly impact inhibitory potency, with CHIR-090, L-161,240, and PF-05081090 showing the highest potency.

How do bacterial resistance mechanisms affect LpxC inhibitor efficacy?

One significant bacterial resistance mechanism affecting LpxC inhibitor efficacy is efflux pump activity. The search results indicate that an efflux pump deficient E. coli strain (W3110 ΔtolC) was eight to ten times more susceptible to LpxC inhibitors compared to the wild type . This suggests that in wild-type bacteria, efflux pumps actively export LpxC inhibitors, reducing their intracellular concentration and effectiveness.

This finding has important implications for inhibitor design and therapeutic strategies:

• Inhibitor optimization: Developing compounds that are poor substrates for efflux pumps could enhance efficacy.

• Combination therapy: Using LpxC inhibitors in combination with efflux pump inhibitors might significantly enhance antimicrobial activity.

• Species specificity: Different bacterial species possess different efflux systems, potentially affecting the relative efficacy of LpxC inhibitors across species.

• Resistance development: Upregulation of efflux pumps represents a potential resistance mechanism that could emerge during treatment with LpxC inhibitors.

Other potential resistance mechanisms not explicitly mentioned in the search results might include:

• Mutations in the lpxC gene affecting inhibitor binding

• Upregulation of lpxC expression to overcome inhibition

• Alternative pathways for outer membrane biogenesis

• Modifications to the bacterial outer membrane reducing inhibitor penetration

How does the metal preference of LpxC relate to bacterial adaptation to different environments?

The dual metal specificity of LpxC, functioning with either Zn(II) or Fe(II), represents a fascinating aspect of metalloenzyme biology with potential implications for bacterial adaptation. The Fe(II)-bound form shows 3-5 fold higher catalytic efficiency compared to the Zn(II) form, and evidence suggests that LpxC can readily switch between these forms .

This metal switching capability may serve as a physiological means of regulating enzyme activity in response to environmental conditions. Several hypotheses can be proposed:

• Adaptation to metal availability: In environments where zinc is limited but iron is abundant, bacteria could maintain LpxC activity by utilizing Fe(II). Conversely, in iron-limited environments, the Zn(II) form would ensure continued function, albeit at reduced efficiency.

• Response to oxidative stress: Iron is susceptible to oxidation, potentially inactivating Fe(II)-LpxC under oxidative conditions. Switching to the more oxidation-resistant Zn(II) form could maintain essential LPS biosynthesis during oxidative stress.

• Metabolic regulation: The difference in catalytic efficiency between Fe(II) and Zn(II) forms could provide a mechanism for fine-tuning LPS production rates in response to environmental or metabolic demands.

• Host-pathogen interactions: During infection, hosts often sequester both zinc and iron as an antimicrobial strategy (nutritional immunity). The ability to use either metal could help pathogenic bacteria overcome this host defense mechanism.

Activity measurements of E. coli cell lysates suggest that the natively expressed enzyme, at least partially, exists as Fe(II)-LpxC , indicating that this form may be physiologically relevant despite the traditional classification of LpxC as a zinc-dependent enzyme.

How does LpxC inhibition affect the balance between LPS and phospholipid biosynthesis?

A key finding from the research on LpxC inhibitors is that they disrupt the balance between lipopolysaccharide (LPS) and phospholipid (PL) biosynthesis. This imbalance appears to be a critical aspect of their antimicrobial mechanism .

Treatment with four of the five tested LpxC inhibitors led to accumulation of lyso-PE, a cleavage product of phosphatidylethanolamine generated by the phospholipase PldA . This suggests that when LPS biosynthesis is blocked by LpxC inhibition, there is a compensatory change in phospholipid metabolism.

The global proteome response to LpxC inhibitor treatment provides further insight into this interconnection. Apart from LpxC itself, FabA and FabB (enzymes responsible for the biosynthesis of unsaturated fatty acids) were consistently upregulated . This indicates that fatty acid biosynthesis, which provides precursors for both LPS and phospholipid pathways, is affected by LpxC inhibition.

This delicate balance between LPS and PL biosynthesis has "great potential as point of attack for antimicrobial intervention" . The interconnection likely exists because:

• Both pathways compete for common precursors like fatty acids

• Proper membrane architecture requires specific ratios of LPS and phospholipids

• Regulatory mechanisms have evolved to coordinate these pathways

Understanding this balance in Synechocystis sp. specifically would require targeted metabolic studies examining how LPS and phospholipid composition changes in response to LpxC inhibition or altered expression.

How can computational approaches enhance LpxC inhibitor discovery?

Computational approaches offer powerful tools for accelerating LpxC inhibitor discovery and optimization. One study successfully applied machine learning techniques to build QSAR models for predicting LpxC inhibitory activity . Several computational strategies can enhance this research area:

• QSAR Modeling: Building on the successful application of Extremely Gradient Boost and Random Forest models with accuracies around 80% , more sophisticated QSAR approaches incorporating 3D information, quantum mechanical parameters, or pharmacophore features could further improve predictive power.

• Structure-Based Design: Utilizing the available structural information on LpxC, such as the solution structure of LpxC from Aquifex aeolicus in complex with the substrate-analog inhibitor TU-514 , to guide rational design through molecular docking and structure-based virtual screening.

• Molecular Dynamics Simulations: Investigating the dynamics of LpxC with different metal cofactors and bound inhibitors to understand subtle differences in binding modes and identify new binding opportunities.

• Network Analysis: Expanding beyond the direct LpxC target to model the broader impact of inhibition on bacterial metabolic networks, potentially identifying synergistic drug targets.

• Activity Cliff Analysis: Further investigating the "consensus activity cliff generators" identified in previous research to understand what structural features lead to large activity differences.

• De Novo Design: Using generative models or fragment-based approaches to design novel chemical scaffolds with optimal properties for LpxC inhibition.

By integrating these computational approaches with experimental validation, researchers can more efficiently develop potent and selective LpxC inhibitors for antimicrobial applications.

What are common challenges in expressing and purifying active recombinant LpxC?

Researchers working with recombinant LpxC may encounter several challenges that can affect protein yield, purity, and activity:

• Metal incorporation: As a metalloenzyme requiring Zn²⁺ or Fe²⁺, ensuring proper metal incorporation is crucial. Complete inhibition by metal-chelating reagents like dipicolinic acid (DPA) and ethylenediaminetetraacetic acid demonstrates LpxC's absolute metal dependency . Supplementing growth media with appropriate metal ions and avoiding chelators during purification is essential.

• Protein stability: LpxC may exhibit limited stability, particularly in the absence of proper metal incorporation. Evidence suggests that inhibitor binding stabilizes LpxC by preventing FtsH-mediated degradation , indicating that inclusion of substrate analogs or inhibitors during purification might enhance stability.

• Expression levels: High-level expression of a metalloenzyme might deplete available metal ions in the expression host, leading to inactive protein. Optimizing induction conditions and metal supplementation can address this issue.

• Proper folding: Lower induction temperatures (16-20°C) typically favor proper folding of complex enzymes like LpxC.

• Activity verification: Developing reliable activity assays is essential, particularly given the marked difference in catalytic efficiency between physiological substrate (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc) and simpler analogs like UDP-GlcNAc (5×10⁶-fold difference in kcat/KM) .

• Metal oxidation: For Fe(II)-LpxC, preventing oxidation during purification by including reducing agents and working under anaerobic conditions may be necessary to maintain activity.

How can the issue of substrate availability for LpxC assays be addressed?

The natural substrate for LpxC, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine, presents a significant challenge for enzymatic studies due to its complex structure and limited commercial availability. Several strategies can address this challenge:

• Chemoenzymatic synthesis: Utilizing upstream enzymes in the lipid A biosynthetic pathway to generate the substrate enzymatically. This approach often yields more stereochemically pure product than chemical synthesis alone.

• Alternative substrates: Using UDP-GlcNAc as a minimal substrate for preliminary studies, while recognizing its substantially lower efficiency (5×10⁶-fold lower kcat/KM) . The large difference in efficiency underscores the importance of the acyl chain for substrate recognition.

• Substrate analogs: Developing simplified substrate analogs that retain key features required for enzyme recognition while being more synthetically accessible.

• Inhibitor-based assays: Leveraging inhibitors like TU-514 for binding studies and competitive assays when direct activity measurement with the natural substrate is not possible.

• Collaborative approaches: Establishing collaborations with synthetic chemistry laboratories specializing in complex carbohydrate synthesis to obtain the natural substrate.

The marked preference for the acylated substrate underscores the importance of the hydrophobic passage in LpxC that accommodates the acyl chain . Any simplified substrates or analogs should ideally retain this feature for meaningful activity measurements.

What strategies can overcome challenges in studying the dual metal specificity of LpxC?

Investigating the dual metal specificity of LpxC presents unique challenges that require specialized approaches:

• Metal-free enzyme preparation: Preparing completely metal-free (apo) LpxC is essential for controlled metal reconstitution studies. This can be achieved through extended dialysis against metal chelators like dipicolinic acid (DPA) , followed by removal of the chelator.

• Anaerobic techniques: For studies with Fe(II)-LpxC, preventing oxidation to Fe(III) is crucial. Working under anaerobic conditions (glove box or Schlenk techniques) and including reducing agents in buffers can maintain the Fe(II) oxidation state.

• Spectroscopic analysis: X-ray absorption spectroscopy (XAS) has been used to reveal differences in coordination number between Fe(II)-LpxC and Zn(II)-LpxC . Other spectroscopic techniques like EPR (for Fe forms) or NMR could provide additional insights into metal coordination.

• Comparative kinetics: Systematic comparison of kinetic parameters (kcat, KM, kcat/KM) between Zn(II)-LpxC and Fe(II)-LpxC under identical conditions can quantify the functional differences between the metal forms.

• Metal switching studies: Investigating the kinetics and thermodynamics of metal exchange between Zn(II) and Fe(II) forms can provide insights into the physiological relevance of metal switching as a regulatory mechanism .

• Protein engineering: Site-directed mutagenesis of metal-coordinating residues can help understand how the protein environment tunes metal selectivity and catalytic properties.

These approaches can help elucidate the molecular basis for the observed 3-5 fold higher catalytic efficiency of Fe(II)-LpxC compared to Zn(II)-LpxC and its potential physiological significance.