Recombinant Geobacter sulfurreducens Shikimate dehydrogenase (aroE)

What is the role of Shikimate dehydrogenase (aroE) in Geobacter sulfurreducens metabolism?

Shikimate dehydrogenase (aroE) in G. sulfurreducens catalyzes the fourth step in the shikimate pathway, which is essential for the biosynthesis of aromatic compounds. This enzyme specifically catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate.

The shikimate pathway is particularly important in G. sulfurreducens as it represents the biosynthetic route for aromatic amino acids and other essential metabolites. Unlike mammals, which lack this pathway, bacteria like G. sulfurreducens rely on it for producing precursors for various cellular components .

In the context of G. sulfurreducens metabolism, aroE functions within a complex network of biosynthetic and energy-generating pathways. The organism possesses a complete TCA cycle that serves to synthesize a diversity of precursor metabolites for biosynthetic reactions . AroE likely interfaces with central carbon metabolism, particularly because the shikimate pathway requires input from both the pentose phosphate pathway and glycolysis.

How does the expression of aroE change under different electron acceptor conditions?

The expression of aroE in G. sulfurreducens appears to be influenced by the electron acceptors available for respiration. This reflects the organism's remarkable metabolic flexibility.

When grown with different electron acceptors such as Fe(III), fumarate, oxygen, or electrodes, G. sulfurreducens exhibits significant changes in its proteome and gene expression patterns. For instance:

• Under Fe(III) reduction conditions, specific cytochromes and other proteins are differentially expressed compared to growth on fumarate or electrodes

• When shifting from anaerobic to microaerobic conditions, G. sulfurreducens demonstrates altered gene expression patterns to adapt to oxygen as an electron acceptor

What methods are recommended for expressing recombinant G. sulfurreducens aroE?

For successful expression of recombinant G. sulfurreducens aroE, researchers should consider the following methodological approach:

Recommended expression system:

• E. coli expression systems have been successfully used for G. sulfurreducens proteins

• Expression plasmids such as pET24b (Novagen) have proven effective for recombinant expression of G. sulfurreducens proteins

Expression protocol:

• Amplify the aroE gene using PCR with primers containing appropriate restriction sites (e.g., NdeI and EcoRI)

• Digest the PCR product with the selected restriction enzymes

• Ligate the fragment into an expression vector (such as pET24b)

• Transform into an appropriate E. coli expression strain

• Induce protein expression with IPTG (typically at 1 mM final concentration)

Purification considerations:

• Include a His-tag for affinity purification

• Consider expressing under microaerobic conditions as used for other G. sulfurreducens proteins

• Verify incorporation of any necessary cofactors through appropriate assays (e.g., spectrophotometric analysis)

A genetic system for G. sulfurreducens has been developed, offering the alternative of homologous expression using IncQ and pBBR1 vectors that can replicate in G. sulfurreducens .

How does G. sulfurreducens aroE compare structurally to homologous enzymes in other organisms?

Structural comparisons between G. sulfurreducens aroE and homologs from other organisms reveal important insights:

While specific structural details for G. sulfurreducens aroE are not directly provided in the search results, we can make informed comparisons based on shikimate dehydrogenase structures from other organisms:

• General architecture: Shikimate dehydrogenases typically display an architecture with two α/β domains separated by a wide cleft, as observed in E. coli AroE and YdiB .

• Cofactor specificity:

  • Most bacterial aroE enzymes exhibit specificity for NADP+ as a cofactor

  • Some organisms possess dual-specificity enzymes that can utilize both NAD+ and NADP+

  • Cofactor specificity is determined by specific residues in the dinucleotide-binding domain

• Substrate binding site:

  • The substrate binding site typically contains conserved residues that interact with 3-dehydroshikimate

  • Conformational changes occur upon substrate binding, with the enzyme switching between open and closed conformations

Based on comparative analysis, G. sulfurreducens aroE likely shares these key structural features while potentially possessing unique adaptations related to its specific metabolic context.

What are the standard assays for measuring Shikimate dehydrogenase activity in G. sulfurreducens extracts?

Standard assays for measuring G. sulfurreducens Shikimate dehydrogenase activity include:

Spectrophotometric assay:

• Prepare cell extracts using buffer systems such as TGED buffer (10 mM Tris-Cl, pH 7.9; 10% glycerol; 0.1 mM EDTA; 0.1 mM DTT) supplemented with protease inhibitors

• Disrupt cells by sonication followed by centrifugation

• Measure aroE activity by monitoring the reduction of NADP+ at 340 nm in the presence of shikimate

• Alternatively, measure the reverse reaction by monitoring the oxidation of NADPH in the presence of 3-dehydroshikimate

Reaction conditions:

• Buffer: 100 mM Tris-HCl, pH 7.5-8.0

• Temperature: 25-30°C

• Substrate concentration: 0.1-1.0 mM shikimate or 3-dehydroshikimate

• Cofactor concentration: 0.1-0.5 mM NADP+ or NADPH

Activity calculation:

• Calculate specific activity as μmol of NADPH formed/oxidized per minute per milligram of protein

• Use an extinction coefficient of 6,220 M-1 cm-1 for NADPH at 340 nm

For more detailed analysis, enzyme fractionation by ammonium sulfate precipitation can be employed, similar to methods used for other G. sulfurreducens proteins .

How does aroE contribute to the adaptation of G. sulfurreducens to different environmental conditions?

The role of aroE in G. sulfurreducens adaptation to different environmental conditions likely extends beyond its primary catalytic function:

Metabolic flexibility:

• G. sulfurreducens demonstrates remarkable metabolic flexibility, adapting to different electron donors and acceptors including acetate, formate, hydrogen, Fe(III), fumarate, and even oxygen under microaerobic conditions

• As part of the shikimate pathway, aroE likely contributes to metabolic adaptation by ensuring continued production of essential aromatic compounds under varying conditions

Oxidative stress response:

• G. sulfurreducens has evolved multiple strategies to deal with oxygen exposure, including complete reduction of oxygen at moderate concentrations and formation of protective layers at higher concentrations

• The shikimate pathway produces precursors for compounds that may play roles in oxidative stress response

• The regulation of aroE expression may be integrated with the organism's stress response systems

Energy conservation:

• G. sulfurreducens adapts its electron transport pathways to maximize energy conservation in response to changes in redox conditions

• AroE, as part of biosynthetic metabolism, would need to coordinate with these energy-generating systems

Environmental significance:

• G. sulfurreducens plays important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells

• Understanding how aroE contributes to adaptation can help optimize these environmental applications

What is the relationship between aroE activity and the electron transport pathways in G. sulfurreducens?

The relationship between aroE activity and electron transport pathways in G. sulfurreducens reveals intricate metabolic integration:

Metabolic intersection:

• Electron transport in G. sulfurreducens is linked to energy generation through the TCA cycle and related pathways

• AroE, while not directly involved in electron transport, relies on reduced cofactors (NADPH) that are generated through cellular metabolism

• This creates a functional dependency between aroE activity and the electron transport status of the cell

Transcriptional coordination:
Research indicates that G. sulfurreducens has evolved sophisticated regulatory systems to coordinate its metabolic activities:

• Gene regulation analysis has identified a transcription factor HgtR that acts as a global regulator for genes involved in biosynthesis and energy generation

• Further research has identified three distinct electron transfer pathways for respiration, with the organism switching between these pathways to adapt to the redox potential of its electron acceptor

Redox balance:

Potential regulatory crosstalk:

• The regulatory networks controlling aroE expression likely intersect with those governing electron transport components

• Network analysis of G. sulfurreducens has identified modules where aroE (GSU2393) interacts with various regulatory influences, potentially linking its expression to electron transport status

What structural and functional adaptations might G. sulfurreducens aroE have compared to homologs from aerobic organisms?

G. sulfurreducens aroE likely exhibits several structural and functional adaptations compared to homologs from aerobic organisms:

Oxygen sensitivity considerations:

• As G. sulfurreducens was initially classified as a strict anaerobe but later shown to tolerate and even use oxygen as an electron acceptor , its aroE might possess adaptations that maintain functionality across varying oxygen concentrations

• Potential adaptations could include more robust redox-sensitive residues or altered surface charges that stabilize the protein structure under varying redox conditions

Cofactor specificity:

• While many shikimate dehydrogenases show specificity for either NAD+ or NADP+, some demonstrate dual specificity

• G. sulfurreducens aroE might exhibit cofactor preferences optimized for its flexible metabolism, potentially favoring NADP+ under standard conditions but retaining some activity with NAD+ for metabolic flexibility

Catalytic efficiency:

• Kinetic parameters of G. sulfurreducens aroE might be optimized for functioning in an organism that frequently experiences electron acceptor limitations

• This could manifest as altered substrate affinity (Km) or catalytic rates (kcat) compared to aerobic counterparts

Structural features:

• While most shikimate dehydrogenases share a common architecture with two α/β domains separated by a catalytic cleft , G. sulfurreducens aroE might feature:

  • Modified substrate-binding pocket architecture

  • Altered conformational dynamics between open and closed states

  • Unique surface properties related to potential protein-protein interactions specific to G. sulfurreducens metabolism

A comparative structural analysis between G. sulfurreducens aroE and homologs from both aerobic and anaerobic organisms would provide valuable insights into these adaptations.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of G. sulfurreducens aroE?

Site-directed mutagenesis offers a powerful approach to investigate the catalytic mechanism of G. sulfurreducens aroE:

Recommended experimental strategy:

• Target residue identification:

  • Use sequence alignment with well-characterized shikimate dehydrogenases to identify conserved catalytic residues

  • Focus on residues in the substrate binding pocket and those involved in cofactor binding

  • Pay particular attention to residues that determine NADP+ vs. NAD+ specificity

• Mutagenesis protocol:

  • Clone the G. sulfurreducens aroE gene into an expression vector such as pET24b

  • Perform site-directed mutagenesis using standard protocols (e.g., QuikChange method)

  • Create single amino acid substitutions (e.g., K→A, D→N) to probe specific catalytic functions

• Expression and purification:

  • Express wild-type and mutant proteins using established protocols for G. sulfurreducens proteins

  • Purify proteins using affinity chromatography (His-tag recommended)

  • Verify protein folding by circular dichroism or fluorescence spectroscopy

• Kinetic analysis:

  • Perform steady-state kinetic analysis measuring both forward and reverse reactions

  • Determine key parameters (kcat, Km) for both wild-type and mutant enzymes

  • Analyze pH-rate profiles to identify potential acid-base catalysts

• Structural characterization:

  • Use X-ray crystallography or cryo-EM to determine structures of wild-type and key mutants

  • Focus on obtaining structures with bound substrates and cofactors

Key residues to target:

• Conserved residues in the catalytic site that interact with the substrate

• Residues that determine cofactor specificity (NADP+ vs. NAD+)

• Residues potentially involved in domain movement during catalytic cycle

Expected outcomes:

• Identification of essential catalytic residues

• Understanding of the structural basis for substrate and cofactor specificity

• Insights into potential adaptations specific to G. sulfurreducens metabolism

What challenges exist in obtaining pure, active recombinant G. sulfurreducens aroE and how can they be addressed?

Obtaining pure, active recombinant G. sulfurreducens aroE presents several challenges that can be addressed through specific methodological approaches:

Challenge 1: Protein solubility

• G. sulfurreducens proteins may have evolved to function optimally under anaerobic conditions

• Solution: Express at lower temperatures (16-20°C) to promote proper folding

• Solution: Include solubility-enhancing fusion tags (MBP, SUMO, or TrxA tags)

• Solution: Optimize induction conditions (lower IPTG concentration, 0.1-0.5 mM)

Challenge 2: Maintaining anaerobic conditions during purification

• AroE may be sensitive to oxidation, affecting its activity or stability

• Solution: Perform purification in an anaerobic chamber

• Solution: Include reducing agents (DTT, β-mercaptoethanol, or TCEP) in all buffers

• Solution: Add oxygen scavengers to buffers (glucose oxidase/catalase system)

Challenge 3: Cofactor retention

• Ensuring the enzyme retains necessary cofactors during purification

• Solution: Supplement purification buffers with small amounts of NADP+

• Solution: Verify cofactor incorporation using spectroscopic methods

Challenge 4: Proteolytic degradation

• G. sulfurreducens aroE may be susceptible to proteolysis

• Solution: Include multiple protease inhibitors in lysis and purification buffers

• Solution: Utilize E. coli strains deficient in key proteases (BL21, Rosetta)

• Solution: Optimize purification speed to minimize time for degradation

Challenge 5: Activity verification

• Ensuring the purified enzyme retains catalytic activity

• Solution: Develop robust activity assays based on NADPH formation/consumption

• Solution: Compare activity with well-characterized homologs from other organisms

• Solution: Optimize assay conditions (pH, temperature, ionic strength)

Purification protocol optimization:

• Cell lysis using TGED buffer (10 mM Tris-Cl, pH 7.9; 10% glycerol; 0.1 mM EDTA; 0.1 mM DTT) supplemented with protease inhibitors

• Initial capture using affinity chromatography (His-tag recommended)

• Further purification using ion exchange chromatography

• Final polishing step using size exclusion chromatography

• Verification of purity by SDS-PAGE and activity using spectrophotometric assays

How does the function of aroE in G. sulfurreducens relate to its remarkable ability to perform extracellular electron transfer?

The function of aroE in G. sulfurreducens may have indirect but significant connections to the organism's extracellular electron transfer (EET) capabilities:

Biosynthetic support for EET machinery:

• G. sulfurreducens possesses an extensive network of c-type cytochromes and other electron transfer proteins essential for EET

• The shikimate pathway, involving aroE, produces precursors for aromatic amino acids and other compounds

• These aromatic compounds may be important structural components of:

  • Cytochromes involved in electron transfer chains

  • Electrically conductive pili (e-pili) that function as "nanowires"

  • Membrane proteins that participate in electron transport

Metabolic integration:

• EET in G. sulfurreducens is closely linked to central metabolism, particularly the TCA cycle

• AroE, as part of the shikimate pathway, connects to central carbon metabolism

• This creates functional dependencies between aroE activity and the energy-generating processes that drive EET

Potential role in adaptation to different electron acceptors:

• G. sulfurreducens can use various electron acceptors including Fe(III) oxides, soluble Fe(III), fumarate, and electrodes

• The organism shows distinct patterns of gene expression and protein abundance depending on the electron acceptor

• AroE expression and activity might be regulated as part of these adaptive responses

Relationship to stress response:

• G. sulfurreducens has evolved mechanisms to deal with oxidative stress

• The shikimate pathway produces precursors for compounds potentially involved in stress protection

• This might be particularly relevant when G. sulfurreducens performs EET under microaerobic conditions

Table: Comparison of G. sulfurreducens growth conditions and potential implications for aroE function

What genomic evidence suggests evolutionary adaptations in G. sulfurreducens aroE compared to related species?

Genomic analysis provides insights into the evolutionary adaptations of G. sulfurreducens aroE compared to related species:

Gene context and organization:

• In G. sulfurreducens, aroE appears to be associated with specific regulatory modules (modules 83 and 121)

• This organization may reflect adaptations to the unique metabolic capabilities of G. sulfurreducens

Phylogenetic considerations:

• G. sulfurreducens belongs to the order Desulfuromonadales, which has undergone specific evolutionary adaptations for extracellular electron transfer

• Similar to how the electrically conductive pili (e-pili) in Geobacter species represent a recent evolutionary innovation , aroE may have undergone adaptations specific to the Geobacter lineage

Potential gene duplication events:

• In some organisms, such as E. coli, there are two shikimate dehydrogenase paralogs (AroE and YdiB)

• Genomic analysis of G. sulfurreducens could reveal whether similar gene duplication events have occurred

• Such duplications often allow functional specialization of enzymes for different metabolic contexts

Horizontal gene transfer considerations:

• The search results indicate that Geobacter species have acquired various genes through horizontal gene transfer

• Analysis of aroE sequence and codon usage could reveal if it was acquired through similar mechanisms

Selective pressures specific to G. sulfurreducens lifestyle:

• As an organism adapted to anaerobic environments with the ability to reduce various electron acceptors , G. sulfurreducens would experience selective pressures distinct from aerobic organisms

• These pressures likely shaped the evolution of all metabolic enzymes, including aroE

• Sequence analysis focusing on positively selected residues could reveal specific adaptations

Comparative genomic approach:

• Align aroE sequences from G. sulfurreducens and related species

• Calculate Ka/Ks ratios to identify sites under positive selection

• Map these sites onto structural models to identify functional implications

• Compare gene neighborhoods across species to identify conserved genetic contexts

Such analysis would provide valuable insights into how aroE has adapted to support the unique metabolic capabilities of G. sulfurreducens.