Recombinant Enterococcus faecalis Ornithine carbamoyltransferase, catabolic (arcB)

What is the function of Ornithine carbamoyltransferase (arcB) in Enterococcus faecalis?

Ornithine carbamoyltransferase (arcB) in E. faecalis is a critical enzyme in the arginine deiminase (ADI) pathway, which enables the bacterium to utilize arginine as an energy source. The enzyme catalyzes the conversion of ornithine and carbamoyl phosphate to citrulline and inorganic phosphate. This reaction is part of a broader metabolic pathway that allows E. faecalis to generate ATP from arginine, particularly under anaerobic conditions or during nutrient limitation.

The ADI pathway shares similarities with the agmatine deiminase pathway in E. faecalis, which also involves a transcarbamylase enzyme that catalyzes an analogous reaction. In the agmatine pathway, ATP is generated from agmatine in three steps, with putrescine transcarbamylase (PTC) performing a similar function to arcB in the arginine pathway . These pathways are particularly important for E. faecalis as they contribute to acid tolerance and provide alternative energy sources, which can be crucial for bacterial survival in various host environments, including during infection processes .

How does arcB differ from other carbamoyltransferases in E. faecalis?

E. faecalis possesses multiple carbamoyltransferases with distinct functions in different metabolic pathways. The catabolic ornithine carbamoyltransferase (arcB) differs from anabolic ornithine carbamoyltransferases in several key aspects:

• Metabolic direction: While anabolic ornithine carbamoyltransferases function in biosynthetic pathways to produce arginine, arcB works in the catabolic direction to degrade arginine for energy production.

• Substrate affinity: arcB has evolved to function optimally in the catabolic direction, with different kinetic parameters compared to anabolic variants.

• Oligomeric structure: Research on related transcarbamylases in E. faecalis, such as putrescine transcarbamylase (PTC), has shown they typically form trimeric structures that likely evolved from ornithine transcarbamylase . This trimeric arrangement is important for catalytic function.

• Substrate specificity: The evolutionary relationship between different carbamoyltransferases is evident in their substrate preferences. For example, studies have shown that PTC can still utilize ornithine as a substrate, albeit poorly, suggesting a common evolutionary origin with arcB .

• Regulatory mechanisms: Expression of arcB is typically induced under different conditions than anabolic variants, reflecting its distinct metabolic role in energy generation rather than biosynthesis.

What expression systems are commonly used for recombinant production of E. faecalis arcB?

Several expression systems have been successfully employed for recombinant production of E. faecalis enzymes, with methodological considerations that apply to arcB production:

• Escherichia coli expression systems:

  • pET vector systems with T7 promoter control in E. coli BL21(DE3) or derivatives

  • Codon optimization for E. coli may be necessary for efficient expression

  • Expression at reduced temperatures (16-25°C) often enhances proper folding of E. faecalis proteins

• Gram-positive expression hosts:

  • Lactococcus lactis expression systems may provide a more suitable environment for proper folding of Gram-positive bacterial proteins

  • The LiAc-DTT method has been successfully used for transformation of L. lactis for expressing E. faecalis proteins

• Fusion protein strategies:

  • Addition of solubility-enhancing tags (His, MBP, SUMO)

  • Careful consideration of tag position and cleavage options

  • Empirical testing of multiple constructs may be necessary to optimize expression

For arcB specifically, the choice between E. coli and Gram-positive hosts should consider factors such as required yield, downstream applications, and the need to maintain native folding and activity. E. coli systems typically provide high yields but may require extensive optimization for proper folding of E. faecalis proteins. In some cases, expressing fragments of the protein separately may be necessary, as demonstrated in studies of other E. faecalis proteins .

What purification strategies yield the highest purity of recombinant arcB?

Purification of recombinant E. faecalis arcB typically employs a multi-step approach to achieve high purity while maintaining enzymatic activity:

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged arcB

  • Careful optimization of imidazole concentration in binding and elution buffers

  • Multiple short affinity columns may yield better results than a single longer column

• Ion exchange chromatography:

  • Anion exchange (e.g., Q Sepharose) based on the predicted pI of arcB

  • Gradient elution to separate closely related contaminants

• Size exclusion chromatography:

  • Final polishing step to separate oligomeric forms and remove aggregates

  • Also provides information about the quaternary structure (expected to be trimeric based on related carbamoyltransferases)

• Stabilizing additives during purification:

  • Addition of glycerol (10-20%) to prevent aggregation

  • Inclusion of reducing agents (DTT, β-mercaptoethanol) if cysteine residues are present

  • Substrate analogs or products may stabilize the enzyme during purification

Research on related E. faecalis enzymes has shown that innovative inhibitors, such as N-(Phosphonoacetyl)-putrescine for PTC, can improve crystallization and potentially be useful during purification by stabilizing the enzyme structure . Similar approaches might be applicable to arcB purification, especially when high purity is required for structural studies.

How can the enzymatic activity of recombinant arcB be measured?

Several methodological approaches can be employed to measure the enzymatic activity of recombinant arcB:

• Colorimetric citrulline detection:

  • The citrulline produced by arcB can be measured using colorimetric assays

  • Reaction of citrulline with diacetyl monoxime and antipyrine produces a colored complex measurable at 464 nm

  • Standard curves using pure citrulline enable quantification

• Coupled enzymatic assays:

  • The phosphate released during the reaction can be measured using coupled enzymatic reactions

  • Malachite green assays for inorganic phosphate detection

  • Continuous spectrophotometric assays using auxiliary enzymes

• Isothermal titration calorimetry (ITC):

  • Direct measurement of heat released during catalysis

  • Also provides binding parameters for substrates and inhibitors

  • Useful for investigating reaction thermodynamics

• High-performance liquid chromatography (HPLC):

  • Direct separation and quantification of reaction substrates and products

  • Particularly useful for determining substrate specificity profiles

  • Can be coupled with mass spectrometry for additional specificity

Typical reaction conditions include 50 mM Tris-HCl buffer (pH 7.5-8.0), 5-10 mM ornithine, 2-5 mM carbamoyl phosphate, and appropriate concentrations of purified enzyme, incubated at 37°C. Activity should be expressed as μmol of citrulline formed per minute per mg of protein, with determination of kinetic parameters (Km, Vmax, kcat) under varying substrate concentrations.

How does the structure of arcB influence its substrate specificity compared to anabolic ornithine carbamoyltransferases?

The structural determinants of substrate specificity in catabolic ornithine carbamoyltransferases like arcB involve subtle but significant differences in the active site architecture and binding pockets. Based on structural studies of related carbamoyltransferases in E. faecalis, several key features likely contribute to the functional specialization of arcB:

• Active site geometry:

  • Catabolic OCTases typically feature altered positioning of catalytic residues that favor the reverse reaction (citrulline to ornithine)

  • Studies of putrescine transcarbamylase (PTC) from E. faecalis have shown that this related enzyme forms a trimeric structure and has evolved from ornithine transcarbamylase

  • The crystal structure of PTC at 1.65 Å resolution has revealed important insights into substrate binding and catalysis that may be applicable to arcB

• Substrate binding pockets:

  • Specific residues in the ornithine/citrulline binding pocket determine substrate preference

  • PTC can utilize ornithine as a substrate, albeit poorly, suggesting evolutionary relationships between these enzymes

• Evolutionary adaptations:

  • Comparative structural analysis suggests that while arcB and anabolic OCTases share core catalytic mechanisms, they have diverged in terms of substrate preference

  • The recruitment of existing enzymes for new metabolic functions appears to be a common theme in the evolution of these pathways

• Inhibitor binding:

  • Studies with N-(Phosphonoacetyl)-putrescine, which strongly inhibits PTC (Ki = 10 nM), provide a model for understanding substrate interactions in related carbamoyltransferases like arcB

  • Similar transition-state analogs could be designed to probe arcB structure and function

Detailed structural comparisons between arcB and anabolic OCTases would reveal specific residues responsible for substrate discrimination, potentially enabling rational engineering of variants with altered specificity or improved catalytic properties.

What role does arcB play in the virulence and stress response of E. faecalis?

The contribution of ornithine carbamoyltransferase (arcB) to E. faecalis virulence and stress response extends beyond its canonical metabolic function:

• Acid stress tolerance:

  • The arginine deiminase pathway, of which arcB is a part, generates ammonia that can neutralize cytoplasmic acidification

  • This acid tolerance mechanism may contribute to E. faecalis survival in acidic host environments such as the urinary tract

• Energy production under stress conditions:

  • The ADI pathway generates ATP through substrate-level phosphorylation, providing energy when oxidative phosphorylation is limited

  • Similar to how the agmatine deiminase pathway enables E. faecalis to make ATP from agmatine in three steps

• Contribution to infection processes:

  • Studies of other E. faecalis enzymes have shown that metabolic adaptations are important for pathogenesis

  • For example, RNases J2, Y, and III mutants are affected in virulence in the Galleria mellonella infection model

  • E. faecalis causes various infections including cystitis, pyelonephritis, catheter-associated UTI, endocarditis, and mixed-organism infections of the abdomen and pelvis

• Response to environmental stresses:

  • Various stress responses in E. faecalis involve specialized enzymes

  • Cold, oxidative, and bile salt stress responses have been linked to specific RNases

  • Similar stress-responsive roles may exist for arcB, particularly under nutrient limitation

• Potential therapeutic target:

  • Understanding arcB's role in virulence could lead to new therapeutic approaches

  • Inhibitors targeting key metabolic enzymes might sensitize E. faecalis to conventional antibiotics

  • The success of dual antibiotic therapy against E. faecalis infections suggests the potential for targeting multiple pathways

Methodological approaches to investigate arcB's role in virulence could include constructing deletion mutants, complementation studies, and evaluation in relevant infection models, similar to approaches used for other E. faecalis virulence determinants .

How do post-translational modifications affect the activity of recombinant arcB?

Post-translational modifications (PTMs) can significantly influence the activity, stability, and regulation of recombinant arcB. Understanding these modifications is crucial for producing enzymatically active protein that accurately reflects the properties of the native enzyme:

• Potential PTMs affecting arcB:

  • Phosphorylation of serine, threonine, or tyrosine residues may alter activity or protein-protein interactions

  • Acetylation of lysine residues can affect catalytic activity and protein stability

  • S-thiolation under oxidative stress conditions may regulate activity in response to the redox state

• Impact on enzymatic parameters:

  • PTMs can alter substrate binding affinity (Km) by inducing conformational changes

  • Catalytic efficiency (kcat/Km) may be enhanced or reduced depending on the modification

  • Oligomerization state and quaternary structure stability might be affected

• Expression system considerations:

  • E. coli expression systems typically lack many of the PTM enzymes present in Gram-positive bacteria

  • Expression in Lactococcus lactis or other Gram-positive hosts might better replicate the native PTM profile

  • Different E. faecalis proteins show varying dependencies on expression systems for proper folding and activity

• Analytical methods for PTM characterization:

  • Mass spectrometry (LC-MS/MS) with appropriate enrichment strategies

  • Phosphoproteomic analysis using TiO2 enrichment or phospho-specific antibodies

  • Activity comparisons between recombinant arcB expressed in different systems

Research on RNA metabolism proteins in E. faecalis has demonstrated that in vitro interactions correlate with physiological roles , suggesting that properly folded and modified recombinant arcB should likewise replicate native functions if appropriate expression and purification strategies are employed.

What are the challenges in obtaining enzymatically active recombinant arcB, and how can they be overcome?

Producing enzymatically active recombinant arcB presents several challenges that require specific strategies to overcome:

• Protein folding and solubility issues:

  • Challenge: Recombinant arcB may form inclusion bodies, particularly in E. coli systems

  • Solutions:

    • Expression at reduced temperatures (16-20°C) to slow folding

    • Use of solubility-enhancing fusion partners (SUMO, MBP, TrxA)

    • Co-expression with molecular chaperones

    • Optimization of induction conditions

• Preserving oligomeric structure:

  • Challenge: arcB likely functions as a trimer (based on related carbamoyltransferases ), and improper assembly affects activity

  • Solutions:

    • Careful optimization of buffer conditions to maintain quaternary structure

    • Size exclusion chromatography to isolate properly assembled oligomers

    • Addition of stabilizing ligands during purification

• Domain-specific expression approaches:

  • Challenge: Full-length arcB may be difficult to express in active form

  • Solutions:

    • Expression of individual domains followed by reconstitution

    • Construction of chimeric proteins with well-folding domains

    • Similar approaches have been successful for other E. faecalis proteins, where domains can be expressed separately

• Alternative expression hosts:

  • Challenge: E. coli may not provide the optimal environment for E. faecalis protein folding

  • Solutions:

    • Expression in Lactococcus lactis using established transformation protocols

    • Optimization of media composition and growth conditions

    • Use of E. faecalis-derived promoters for expression in related organisms

• In vitro refolding strategies:

  • Challenge: Recovering active enzyme from inclusion bodies

  • Solutions:

    • Stepwise dialysis with decreasing denaturant concentrations

    • Addition of chemical chaperones during refolding

    • Pulse refolding techniques to prevent aggregation

A systematic approach testing multiple expression constructs, hosts, and conditions will typically be necessary to identify optimal conditions for producing active recombinant arcB.

How can site-directed mutagenesis be used to enhance the catalytic efficiency of arcB?

Site-directed mutagenesis offers a powerful approach to enhance the catalytic properties of arcB for research or biotechnological applications. A systematic mutagenesis strategy would include:

• Rational design targets based on structural knowledge:

  • Active site residues involved in substrate binding and catalysis

  • Second-shell residues that influence active site geometry

  • Subunit interface residues that affect quaternary structure and cooperativity

  • Analysis of putrescine transcarbamylase (PTC) structure provides a useful model

• Specific strategies to enhance catalytic efficiency:

  • Modifying substrate binding pocket residues to improve Km

  • Targeting catalytic residues to enhance turnover rate (kcat)

  • Engineering pH optimum by altering pKa of key residues

  • Introducing stabilizing interactions to improve thermostability

• Methodological approach:

  • Homology modeling of arcB based on related carbamoyltransferases

  • Molecular dynamics simulations to identify residues with high mobility

  • QuikChange or Gibson Assembly methods for mutagenesis

  • High-throughput screening assays to evaluate multiple variants efficiently

• Case study example:

  • The successful crystallization and characterization of E. faecalis PTC forming a covalent adduct with an agmatine-derived amidine reactional intermediate provides insights that could guide arcB mutagenesis

  • N-(Phosphonoacetyl)-putrescine has been shown to strongly inhibit PTC (Ki = 10 nM), providing a model for transition state interactions

• Mutation categories to consider:

  • Conservative mutations that maintain chemical properties but fine-tune interactions

  • Non-conservative mutations that introduce new functional properties

  • Multiple mutations to capture synergistic effects

A systematic mutagenesis campaign would evaluate variants using steady-state kinetics to determine changes in Km, kcat, and kcat/Km, as well as substrate specificity profiles and stability under various conditions relevant to E. faecalis pathophysiology.

What are the implications of arcB polymorphisms in different E. faecalis strains for antimicrobial resistance?

Genetic variations in arcB across different E. faecalis strains may have significant implications for antimicrobial resistance through both direct and indirect mechanisms:

• Direct effects on stress tolerance:

  • Polymorphisms affecting arcB activity may enhance acid tolerance

  • Improved energy generation through the ADI pathway could support survival during antibiotic exposure

  • E. faecalis is known to cause a variety of infections and shows variable antibiotic susceptibility patterns

• Strain-specific considerations:

  • E. faecalis is more frequently retrieved from sites of infection compared to other enterococci

  • Different strains may contain specific arcB variants associated with enhanced pathogenicity

  • E. faecium is more likely to be resistant to commonly used antibiotics such as ampicillin, suggesting metabolic differences between species matter

• Methodological approaches to investigate arcB polymorphisms:

  • PCR amplification and sequencing of arcB from diverse clinical isolates

  • Recombinant expression of variant arcB proteins to assess enzymatic properties

  • Construction of isogenic strains with arcB variants through allelic exchange methods similar to those used for other E. faecalis genes

• Potential mechanisms linking arcB to antimicrobial resistance:

  • Metabolic adaptation: Enhanced arginine catabolism may compensate for antibiotic-induced metabolic stress

  • Biofilm formation: Metabolic pathways influence biofilm development and associated resistance

  • Energy production: ATP generation through the ADI pathway could support resistance mechanisms requiring energy

• Treatment implications:

  • Dual antibiotic therapy with a cell-wall active agent plus a synergistic agent is necessary when treating serious enterococcal infections

  • Understanding metabolic adaptations could lead to more effective combination therapies

  • Infectious diseases consultation may be beneficial for resistant or refractory infections

Research methodologies similar to those used for studying other E. faecalis virulence factors and stress responses could elucidate the relationship between arcB variants and antimicrobial resistance .

How can structural studies of arcB inform the design of specific inhibitors?

Structural characterization of arcB provides a foundation for rational inhibitor design, potentially leading to novel therapeutics targeting E. faecalis infections:

• Structural determination approaches:

  • X-ray crystallography of purified recombinant arcB

  • Homology modeling based on related structures, such as putrescine transcarbamylase from E. faecalis

  • Cryo-electron microscopy for structure determination in different functional states

• Critical structural features for inhibitor design:

  • Active site architecture and catalytic residues

  • Substrate binding pockets and specificity-determining regions

  • Allosteric sites that might influence enzyme activity

  • Subunit interfaces in the trimeric structure (based on related carbamoyltransferases)

• Inhibitor design strategies:

  • Transition state analogs that mimic the reaction intermediate

  • Studies with N-(Phosphonoacetyl)-putrescine have shown strong inhibition (Ki = 10 nM) of the related PTC enzyme

  • Similar phosphonoacetyl derivatives could be designed for arcB

  • The successful crystallization of PTC with this inhibitor suggests a similar approach could work for arcB

• Structure-based computational approaches:

  • Virtual screening of compound libraries against the arcB structure

  • Molecular dynamics simulations to identify transient binding pockets

  • Fragment-based drug design targeting multiple binding sites

• Validation and optimization pipeline:

  • In vitro enzymatic assays with purified recombinant arcB

  • Structure-activity relationship studies to optimize potency and selectivity

  • Evaluation in cellular systems to confirm target engagement

  • Assessment of effects on E. faecalis virulence and antimicrobial susceptibility

Targeted inhibition of arcB could potentially sensitize E. faecalis to conventional antibiotics or reduce virulence, offering a new strategy to combat infections caused by this opportunistic pathogen, which is increasingly recognized as a significant human pathogen .

What are the discrepancies between in vitro and in vivo studies of arcB function, and how can they be reconciled?

The translation of in vitro findings about arcB to its in vivo function often reveals discrepancies that require careful consideration and integrated approaches to resolve:

• Common discrepancies observed:

  • Substrate affinities measured in vitro may not reflect intracellular conditions

  • Regulatory mechanisms observed in cell extracts might differ from those in intact cells

  • Protein-protein interactions that occur in vivo may be disrupted during purification

  • Post-translational modifications present in vivo may be absent in recombinant systems

• Factors contributing to discrepancies:

  • Intracellular environment (pH, ionic strength, macromolecular crowding)

  • Metabolic context and substrate availability in different growth conditions

  • Interactions with other enzymes in the arginine deiminase pathway

  • Similar discrepancies have been observed in studies of other E. faecalis metabolic pathways

• Methodological approaches to reconcile differences:

  • Construction of reporter strains to monitor arcB activity in living cells

  • Metabolic flux analysis to measure arginine catabolism in different conditions

  • Systematic comparison of arcB mutant phenotypes with biochemical properties

  • Methods for gene deletion and complementation in E. faecalis have been well-established

• Integrative strategies:

  • Combining transcriptomics, proteomics, and metabolomics to create a systems-level view

  • Studies of RNA metabolism proteins in E. faecalis demonstrated that in vitro interactions correlate with physiological roles

  • Mutation analysis in infection models such as Galleria mellonella can link biochemical properties to pathogenesis

• Practical considerations for researchers:

  • Design experiments that bridge in vitro and in vivo conditions

  • Validate findings across multiple experimental systems

  • Consider the physiological context of E. faecalis in different host environments

  • E. faecalis can cause various infections with different metabolic environments

By systematically addressing these discrepancies through complementary approaches, researchers can develop a more complete understanding of arcB function in its native context and better translate biochemical findings to physiological and pathological relevance in E. faecalis infections.