Recombinant Enterococcus faecalis Putative agmatine deiminase (aguA)

What is agmatine deiminase (AguA) and what is its role in Enterococcus faecalis metabolism?

• Agmatine → Carbamoylputrescine + NH₃ (catalyzed by AguA)

• Carbamoylputrescine + Pi → Putrescine + Carbamoylphosphate (catalyzed by AguB)

• Carbamoylphosphate + ADP → ATP + CO₂ + NH₃ (catalyzed by AguC)

How is the aguA gene organized within the E. faecalis genome?

The aguA gene is part of a gene cluster dedicated to agmatine catabolism in E. faecalis. The complete locus is organized as an operon consisting of:

• aguR: Encodes a transcriptional regulator of the LuxR family located in a divergent orientation to the metabolic genes

• aguB: Encodes putrescine transcarbamylase

• aguD: Encodes the agmatine/putrescine antiporter

• aguA: Encodes agmatine deiminase

• aguC: Encodes carbamate kinase
  These genes are transcriptionally organized with aguBDAC in a single operon, transcribed from the aguB promoter (PaguB), while aguR has its own promoter (PaguR) . This genetic architecture is conserved in E. faecalis and displays a high degree of identity to that found in Streptococcus mutans, although the regulation differs between the species .

What expression systems are available for recombinant production of E. faecalis AguA?

Two primary expression systems have been successfully employed for the recombinant production of E. faecalis AguA:

• Heterologous expression in E. coli:
  The aguA gene (EF0734/agcA) from E. faecalis has been successfully cloned, overexpressed in E. coli, and the recombinant protein purified to homogeneity . This approach typically involves:

  • PCR amplification of the aguA coding sequence

  • Cloning into an E. coli expression vector (such as pET series)

  • Transformation into a suitable E. coli strain (such as BL21(DE3))

  • Induction of expression using IPTG

  • Purification via affinity chromatography when expressed with a tag

• Homologous expression in E. faecalis using the agmatine-inducible system:
  The pAGEnt vector system has been developed specifically for E. faecalis, which combines:

  • The aguR inducer gene

  • The aguB promoter (PaguB)

  • A multiple cloning site

  • A C-terminal 10-amino-acid His-tag for purification
    Using the pAGEnt system, expression can be induced by adding agmatine to the culture medium, with a close correlation between agmatine concentration and expression levels .

What are the optimal conditions for expressing and purifying functional recombinant AguA?

For optimal expression and purification of functional recombinant AguA, the following methodological approach is recommended:

• Expression in E. coli:

  • Use a strain such as BL21(DE3) for high-level expression

  • Culture at 37°C until OD₆₀₀ reaches 0.6-0.8

  • Induce with 0.5-1 mM IPTG

  • Lower temperature to 25-30°C after induction to enhance protein folding

  • Continue expression for 4-6 hours or overnight

• For expression in E. faecalis using pAGEnt:

  • Culture E. faecalis to mid-log phase

  • Induce with 60 mM agmatine for optimal expression

  • Harvest after 3-4 hours of induction

• Purification strategy:

  • Lyse cells by sonication or French press in a buffer containing:

    • 50 mM Tris-HCl (pH 8.0)

    • 300 mM NaCl

    • 10 mM imidazole

    • Protease inhibitors

  • Clarify lysate by centrifugation (20,000 × g, 30 min)

  • Purify His-tagged AguA using Ni-NTA affinity chromatography

  • Elute with an imidazole gradient (50-250 mM)

  • Further purify by size exclusion chromatography if higher purity is required

  • Assess purity by SDS-PAGE and activity by enzyme assays
    The crystal structure of AgDI has been determined at 1.65 Å resolution, showing the enzyme forming a covalent adduct with an agmatine-derived amidine reactional intermediate . This structural information can be valuable for assessing protein quality and designing mutagenesis studies.

What methods are used to measure AguA enzymatic activity?

Several methods have been established to measure AguA enzymatic activity:

• Ammonia production assay:

  • This direct assay measures the ammonia released during the conversion of agmatine to carbamoylputrescine

  • The reaction mixture typically contains:

    • 50 mM potassium phosphate buffer (pH 7.0)

    • 1-10 mM agmatine

    • Purified AguA enzyme

  • After incubation (typically 30 minutes at 37°C), the reaction is stopped

  • Ammonia can be quantified using:

    • Nessler's reagent (colorimetric)

    • Glutamate dehydrogenase-coupled assay (spectrophotometric)

    • Ion-selective electrodes

    • High-performance liquid chromatography (HPLC)

• Carbamoylputrescine detection:

  • Measures the product carbamoylputrescine directly

  • Can be performed using HPLC or LC-MS/MS

  • Requires appropriate standards for quantification

• Coupled enzyme assay:

  • Links AguA activity to putrescine transcarbamylase and carbamate kinase reactions

  • Measures ATP production spectrophotometrically using auxiliary enzymes (hexokinase and glucose-6-phosphate dehydrogenase)

  • Allows for continuous monitoring of activity

What are the kinetic parameters of recombinant E. faecalis AguA?

Based on published studies, the kinetic parameters of recombinant E. faecalis AguA include:

How is expression of the aguA gene regulated in E. faecalis?

Expression of aguA in E. faecalis is regulated through multiple mechanisms:

• Agmatine-dependent induction:

  • The presence of exogenous agmatine induces expression of the aguBDAC operon, including aguA

  • This induction is mediated by AguR, a transcriptional regulator that responds to agmatine

  • AguR activates transcription from the aguB promoter in the presence of agmatine

• Carbon catabolite repression (CCR):

  • The AgDI system in E. faecalis is subject to carbon catabolite repression

  • CCR operates via a mechanism involving:

    • CcpA (catabolite control protein A)

    • P-Ser-HPr (phosphorylated form of HPr)

    • A cre site (catabolite-responsive element) located 55 nt upstream of the +1 position of the aguB promoter

• Mannose phosphotransferase system (PTS Man):

  • Components of the PTS Man system also contribute to CCR in E. faecalis

  • Complete relief of PTS-sugar repressive effects is observed only in a PTS Man and CcpA double-defective strain

• pH independence:

  • Unlike the homologous AgDI system in Streptococcus mutans, the aguBDAC operon in E. faecalis is not induced in response to low pH

  • This suggests that regulation has evolved differently in E. faecalis to adapt to its gastrointestinal niche

What experimental approaches can be used to study the regulation of aguA expression?

Several experimental approaches can be employed to study the regulation of aguA expression:

• Reporter gene assays:

  • Fusion of the aguB promoter region to reporter genes (e.g., gfp, lacZ)

  • Measurement of reporter activity under various conditions:

    • Different agmatine concentrations

    • Various carbon sources

    • pH variations

    • Growth phases

• Real-time quantitative PCR (RT-qPCR):

  • Direct measurement of aguA transcript levels

  • Comparative analysis of expression in wildtype and regulatory mutants (ΔaguR, ΔccpA)

  • Evaluation of expression kinetics in response to agmatine addition

• Electrophoretic mobility shift assays (EMSA):

  • Investigation of AguR binding to the aguB promoter region

  • Analysis of CcpA binding to the cre site

  • Determination of binding affinities and specificity

• DNase I footprinting:

  • Identification of precise binding sites for regulatory proteins on the aguB promoter

  • Mapping of protected regions under different conditions

• Chromatin immunoprecipitation (ChIP):

  • In vivo analysis of regulatory protein binding to promoter regions

  • Identification of genome-wide binding sites for AguR or CcpA
    These experimental approaches can provide comprehensive insights into the molecular mechanisms governing aguA expression in E. faecalis.

How does the AgDI pathway contribute to E. faecalis physiology and pathogenesis?

The AgDI pathway, with AguA as a key enzyme, contributes to several aspects of E. faecalis physiology and potential pathogenesis:

• Energy generation:

  • The AgDI pathway generates 1 ATP per agmatine molecule processed

  • This provides an alternative energy source, particularly important under nutrient-limited conditions

  • ATP production contributes to bacterial growth and persistence in various environments

• pH homeostasis and acid resistance:

  • The release of ammonia during agmatine catabolism helps neutralize external medium acidity

  • This contributes to E. faecalis survival in acidic environments, although the pathway is not directly pH-induced like in other bacteria

  • Enhanced bacterial growth is observed due to this pH neutralization effect

• Polyamine metabolism:

  • The AgDI pathway results in putrescine production

  • Putrescine is the polyamine most commonly detected in dairy products and can affect food quality

  • Polyamines play roles in bacterial growth, biofilm formation, and stress responses

• Virulence regulation:

  • While not directly identified as a virulence factor, components of polyamine metabolism have been implicated in enterococcal pathogenesis

  • E. faecalis virulence involves multiple factors, including surface adhesion substances and hemolysins, which may be influenced by metabolic capabilities

• Ecological adaptation:

  • The regulation of the aguBDAC operon in E. faecalis has evolved to obtain energy and resist stressful conditions

  • This adaptation helps E. faecalis persist and colonize gastrointestinal niches

How does AguA activity differ from other agmatine-processing enzymes in bacteria?

AguA (agmatine deiminase) differs from other agmatine-processing enzymes in several key aspects:

• Comparison with agmatinase:

  • Agmatinase converts agmatine directly to putrescine and urea

  • AguA converts agmatine to carbamoylputrescine and ammonia

  • Agmatinase is a metalloenzyme dependent on Mn²⁺, while AguA is not metal-dependent

  • These represent two distinct pathways for agmatine catabolism in bacteria

• Structural and catalytic differences:

  • AguA from E. faecalis exhibits 54% sequence identity with the AgDI encoded by the aguA gene of Pseudomonas aeruginosa

  • It shows only 11.6% identity with arginine deiminase (ADI) from E. faecalis, despite catalyzing a similar reaction with a different substrate

  • The crystal structure of AguA reveals a covalent adduct with an agmatine-derived amidine reactional intermediate, providing insights into its catalytic mechanism

• Inhibition profiles:

  • Metformin has been identified as a competitive inhibitor of bacterial agmatinase with a Ki of 1 mM

  • Metformin analogs (phenformin, buformin, and galegine) are even more potent inhibitors

  • In contrast, the effect of these compounds on AguA activity remains to be fully characterized

• Evolutionary aspects:

  • The aguA gene appears to be present predominantly in oral and gastrointestinal microorganisms

  • This suggests specific adaptation to these ecological niches

  • The gene context analysis reveals conservation in certain bacterial groups, indicating horizontal gene transfer events may have occurred

How can recombinant AguA be used as a tool for studying bacterial metabolism?

Recombinant AguA can serve as a valuable tool for studying bacterial metabolism in several advanced applications:

What structural and functional investigations have revealed insights into AguA's catalytic mechanism?

Advanced structural and functional investigations have provided significant insights into AguA's catalytic mechanism:

• Crystal structure analysis:

  • The 1.65 Å resolution structure of AguA has been determined showing a covalent adduct with an agmatine-derived amidine reactional intermediate

  • This structure reveals the architecture of the active site and substrate binding pocket

  • A catalytic cysteine residue (Cys357 in AguA) is conserved in both agmatine deiminases and arginine deiminases and plays a key catalytic role

• Reaction mechanism:

  • The catalytic mechanism likely involves nucleophilic attack by the conserved cysteine on the guanidinium carbon of agmatine

  • Formation of a tetrahedral intermediate, followed by collapse to form a thioester

  • Subsequent hydrolysis of the thioester releases carbamoylputrescine and regenerates the enzyme

• Site-directed mutagenesis studies:

  • Mutation of the catalytic cysteine abolishes enzymatic activity

  • Other conserved residues in the active site have been identified that participate in substrate binding and catalysis

  • These studies help distinguish the roles of specific amino acids in the reaction mechanism

• Substrate specificity:

  • Structure-function analyses reveal why AguA preferentially acts on agmatine rather than similar compounds like arginine

  • The active site geometry is optimized for accommodating the agmatine structure

  • Understanding these specificity determinants can guide engineering efforts for altered substrate specificities

• Inhibitor studies:

  • Analysis of enzyme-inhibitor complexes provides insights into transition state stabilization

  • Identification of key interactions that could be targeted for drug design

  • Development of transition state analogs as potential enzyme inhibitors

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

Researchers commonly encounter several challenges when expressing and purifying active recombinant AguA:

• Inclusion body formation:

  • Problem: Overexpression in E. coli often leads to inclusion body formation

  • Solution:

    • Lower the induction temperature (16-25°C)

    • Reduce IPTG concentration (0.1-0.5 mM)

    • Use E. coli strains optimized for difficult protein expression (e.g., Rosetta, Arctic Express)

    • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

    • Use fusion tags that enhance solubility (MBP, SUMO)

• Low enzymatic activity:

  • Problem: Purified enzyme shows lower than expected activity

  • Solution:

    • Include stabilizing agents in purification buffers (glycerol, DTT)

    • Check for metal ion requirements or inhibitors in buffers

    • Optimize storage conditions to prevent activity loss

    • Verify proper folding using circular dichroism or fluorescence spectroscopy

• Proteolytic degradation:

  • Problem: Enzyme undergoes degradation during expression or purification

  • Solution:

    • Use protease-deficient E. coli strains

    • Include protease inhibitors during purification

    • Minimize handling time and maintain low temperatures

    • Consider adding stabilizing agents or optimizing buffer conditions

• Contaminating activities:

  • Problem: Preparations contain other enzymes that interfere with activity assays

  • Solution:

    • Implement additional purification steps (ion exchange, size exclusion)

    • Design specific activity assays that distinguish AguA activity

    • Use negative controls (heat-inactivated enzyme, catalytic mutants)

• Expression in E. faecalis:

  • Problem: Genetic manipulation of E. faecalis is challenging due to restriction modification systems

  • Solution:

    • Use E. faecalis strains with defective restriction systems

    • Modify plasmid DNA to match E. faecalis methylation patterns

    • Consider electroporation optimization protocols specific for E. faecalis

How can researchers optimize enzyme assays for reliable measurement of AguA activity?

To optimize enzyme assays for reliable measurement of AguA activity, researchers should consider the following methodological approaches:

• Assay validation:

  • Establish linear range of the assay with respect to:

    • Enzyme concentration

    • Reaction time

    • Substrate concentration

  • Determine detection limits and sensitivity

  • Validate with appropriate positive and negative controls

• Reaction conditions optimization:

  • Systematically test:

    • pH range (typically 6.0-8.0)

    • Temperature (25-45°C)

    • Buffer composition (phosphate, HEPES, Tris)

    • Ionic strength

  • Create a standardized protocol to ensure reproducibility

• Interference elimination:

  • Test for potential interfering substances in enzyme preparations

  • Include appropriate blanks and controls

  • Consider using specific inhibitors to confirm activity is due to AguA

• Detection method selection:

  • For ammonia detection, compare methods:

    • Nessler's reagent: simple but less sensitive

    • Glutamate dehydrogenase-coupled assay: more sensitive but complex

    • Ion-selective electrodes: real-time but equipment-intensive

    • Indophenol blue method: sensitive colorimetric assay

  • For LC-MS/MS methods:

    • Develop specific MRM transitions for agmatine and carbamoylputrescine

    • Use isotopically labeled internal standards

    • Optimize chromatographic separation

• Data analysis:

  • Apply appropriate kinetic models (Michaelis-Menten, allosteric)

  • Use statistical methods to evaluate reproducibility

  • Account for background rates and spontaneous reactions

  • Consider using non-linear regression for parameter estimation
    A standardized protocol might include:

• Reaction mixture: 50 mM HEPES buffer (pH 7.5), 1 mM agmatine, purified AguA (0.1-1 μg)

• Incubation: 37°C for 15-30 minutes

• Reaction termination: Boiling for 5 minutes or addition of TCA (final 5%)

• Ammonia quantification: Glutamate dehydrogenase-coupled assay measuring NADH oxidation at 340 nm

• Controls: Enzyme-free and substrate-free reactions

How does E. faecalis AguA compare with homologous enzymes from other bacterial species?

E. faecalis AguA shares similarities and differences with homologous enzymes from other bacterial species:

What evolutionary insights can be gained from studying the agmatine deiminase pathway across bacterial species?

Studying the agmatine deiminase pathway across bacterial species provides several evolutionary insights:

• Horizontal gene transfer:

  • Gene context analysis reveals that aguR is present in oral and gastrointestinal microorganisms

  • This suggests potential horizontal transfer events between bacteria sharing these niches

  • The conservation of the entire operon structure across certain bacterial lineages supports this hypothesis

• Adaptive evolution:

  • The AgDI pathway appears to have evolved differently in various bacteria to suit their ecological niches:

    • In E. faecalis, regulation has evolved to obtain energy and resist conditions in gastrointestinal environments

    • In S. mutans, the pathway is critical for acid resistance in the oral cavity

    • In other bacteria, the pathway may serve different primary functions

• Functional diversification:

  • AguA and arginine deiminase (ADI) share structural similarities but act on different substrates

  • The minimal sequence identity (11.6%) between E. faecalis AguA and ADI suggests ancient divergence

  • The conserved catalytic mechanism but divergent substrate specificity illustrates functional specialization

• Metabolic integration:

  • The AgDI pathway intersects with polyamine metabolism, which is fundamental to cellular functions

  • The evolution of regulatory mechanisms that coordinate polyamine homeostasis reflects the importance of these compounds

  • The connection to ATP generation suggests the pathway may have been selected for during energy-limited conditions

• Host-microbe coevolution:

  • Agmatine is a bioactive compound in mammals with effects on metabolism and neural function

  • Bacterial metabolism of agmatine may influence host physiology

  • Recent findings that metformin inhibits bacterial agmatinase suggest potential roles in host-microbe interactions and drug effects
    Understanding these evolutionary patterns can inform approaches to targeting the pathway for therapeutic purposes or harnessing it for biotechnological applications.

What are emerging research areas involving recombinant E. faecalis AguA?

Several exciting research frontiers are emerging around recombinant E. faecalis AguA:

• Drug development targeting polyamine metabolism:

  • Recent discovery that metformin inhibits bacterial agmatinase has opened new avenues for investigating how drugs targeting agmatine metabolism might affect host-microbe interactions

  • Structure-based design of specific inhibitors for AguA could lead to novel antimicrobials

  • Understanding differences between bacterial and mammalian enzymes could enable selective targeting

• Metabolic engineering applications:

  • Using the agmatine-inducible expression system (pAGEnt) for controlled protein production in E. faecalis

  • Engineering metabolic pathways to channel agmatine processing for biotechnological purposes

  • Development of E. faecalis strains with enhanced or modified agmatine metabolism

• Microbiome research:

  • Investigating the role of AguA and the AgDI pathway in gut microbiome dynamics

  • Understanding how E. faecalis agmatine metabolism affects host metabolic functions

  • Exploring connections between AgDI activity and host diseases (metabolic disorders, inflammatory conditions)

• Synthetic biology tools:

  • Development of genetic circuits using the agmatine-responsive elements

  • Creation of biosensors for detection of agmatine in complex environments

  • Engineering cellular response systems based on agmatine detection

• Structural biology advances:

  • Application of cryo-EM to study AguA in complex with other pathway enzymes

  • Investigation of protein dynamics during catalysis using techniques like NMR or hydrogen-deuterium exchange

  • Computational approaches to understand substrate specificity and enzyme evolution

What methodological advances are needed to address current research gaps?

Several methodological advances would help address current research gaps related to recombinant E. faecalis AguA:

• Improved genetic tools for E. faecalis:

  • Development of more efficient transformation methods for clinical isolates

  • CRISPR-Cas9 systems optimized for E. faecalis genetic manipulation

  • Strategies to overcome restriction modification barriers that limit genetic manipulation

  • Methods to introduce large DNA fragments or multiple genetic modifications simultaneously

• Advanced analytical techniques:

  • Development of high-throughput screening methods for AguA inhibitors

  • Improved methods for measuring intracellular agmatine and putrescine levels

  • Metabolomic approaches to track nitrogen flux through the AgDI pathway

  • Single-cell techniques to monitor AguA activity in mixed bacterial populations

• In vivo models and approaches:

  • Development of animal models to study E. faecalis agmatine metabolism in vivo

  • Methods to track bacterial gene expression in the gastrointestinal tract

  • Techniques to manipulate bacterial metabolism without affecting colonization

  • Approaches to study host-microbe metabolic interactions

• Computational and systems biology tools:

  • Integration of multi-omics data to understand the role of AguA in bacterial physiology

  • Machine learning approaches to predict AguA substrate specificity and inhibitor binding

  • Metabolic modeling of E. faecalis to predict the impact of AgDI pathway perturbations

  • Network analysis to understand regulatory connections to other metabolic pathways

• Structural and biophysical methods:

  • Time-resolved structural studies to capture enzyme intermediates

  • Methods to study protein-protein interactions between AguA and other pathway components

  • Biophysical techniques to measure binding kinetics with potential inhibitors

  • Approaches to visualize enzyme localization within bacterial cells Addressing these methodological challenges will advance our understanding of AguA's role in bacterial physiology and could lead to novel applications in biotechnology and medicine.