Recombinant Geobacter sulfurreducens 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is the function of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) in Geobacter sulfurreducens?

DXR (encoded by gene Rv2870c in Geobacter sulfurreducens) catalyzes the NADP-dependent rearrangement and reduction of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) in the methylerythritol phosphate (MEP) pathway . This represents the second and rate-limiting step in the MEP pathway, which is essential for isoprenoid biosynthesis. The enzyme catalyzes a two-step reaction: first, a Mg²⁺-triggered rearrangement of DXP into a non-isolable aldehyde intermediate, followed by an NADPH-dependent reduction to form MEP . This reaction is particularly significant as it represents the first committed step in isoprenoid biosynthesis, as DXP is also involved in the biosynthesis of vitamins B₁ and B₆ .

What are the optimal conditions for expressing recombinant Geobacter sulfurreducens DXR?

Based on similar recombinant protein expression approaches for G. sulfurreducens proteins, the following methodology is recommended:

• Expression System: Escherichia coli BL21(DE3) or similar expression strain that provides tight control of expression.

• Vector Selection: pET-based vectors containing T7 promoter systems have shown efficacy for expressing recombinant G. sulfurreducens proteins, as demonstrated with cytochrome c₇ .

• Growth Conditions:

  • Medium: LB or 2×YT supplemented with appropriate antibiotics

  • Temperature: Initial growth at 37°C until OD₆₀₀ reaches 0.6-0.8, followed by induction at lower temperatures (16-25°C) to enhance soluble protein expression

  • Induction: 0.1-0.5 mM IPTG, with expression continued for 16-20 hours at reduced temperature

• Co-expression Considerations: When expressing proteins requiring post-translational modifications (as seen with cytochromes), co-expression with required maturation genes may be necessary .

For optimal expression of functional DXR, supplementation with cofactors (NADPH and Mg²⁺) during cell lysis and purification steps is recommended to maintain structural integrity.

What purification strategies are most effective for obtaining high-purity recombinant Geobacter sulfurreducens DXR?

A multi-step purification process is recommended:

Typical yields of 5-10 mg of purified protein per liter of bacterial culture can be expected, comparable to yields reported for other G. sulfurreducens recombinant proteins .

How can the catalytic activity of recombinant Geobacter sulfurreducens DXR be measured?

DXR activity can be measured using several complementary approaches:

• Spectrophotometric NADPH Oxidation Assay:

  • Principle: Monitoring the decrease in NADPH absorbance at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Reaction mixture: 100 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, 0.15 mM NADPH, 0.5-2 mM DXP, and purified enzyme

  • Temperature: Typically performed at 30°C

  • Calculate activity using the equation:
    Activity (μmol/min/mg) = (ΔA₃₄₀/min × reaction volume)/(6.22 × enzyme amount × light path)

• LC-MS Based Product Detection:

  • More sensitive approach for detecting the MEP product formation

  • Requires quenching the reaction with methanol followed by LC-MS analysis

  • Enables direct quantification of the MEP product formed

• Coupled Enzyme Assay:

  • Can be used when direct measurement is challenging

  • Couples the formation of MEP to a secondary reaction that generates a detectable signal

Typical kinetic parameters for bacterial DXR enzymes include K_m values for DXP in the range of 50-300 μM and k_cat values of 1-10 s⁻¹, though specific values for G. sulfurreducens DXR should be determined experimentally.

What factors influence the activity of recombinant Geobacter sulfurreducens DXR?

Several factors significantly impact DXR activity:

• Metal Ion Requirement:

  • Mg²⁺ is essential for enzymatic activity

  • Other divalent cations (Mn²⁺, Co²⁺) may support activity but usually with reduced efficiency

  • Optimal Mg²⁺ concentration is typically 1-5 mM

• pH Optimum:

  • Most bacterial DXR enzymes show optimal activity at pH 7.5-8.0

  • Activity decreases significantly below pH 6.5 or above pH 8.5

• Temperature Effects:

  • G. sulfurreducens grows optimally at 30°C, suggesting the enzyme may be most active near this temperature

  • Enzyme stability decreases significantly above 40°C

• Redox Conditions:

  • Activity is sensitive to oxidizing conditions that can affect critical cysteine residues

  • Reducing agents (DTT, β-mercaptoethanol) at 1-5 mM help maintain activity

• Phosphate Sensitivity:

  • High phosphate concentrations (>50 mM) can inhibit activity by competing with substrate binding

  • Use Tris-HCl or HEPES buffers instead of phosphate buffers for activity assays

When designing experiments to measure DXR activity, consider that G. sulfurreducens can grow under microaerobic conditions with a maximum specific oxygen uptake rate of 95 mg O₂ g CDW⁻¹ h⁻¹ , which may affect enzyme stability during cell growth and protein purification.

How does Geobacter sulfurreducens DXR participate in electron transfer mechanisms relevant to bioremediation?

While DXR itself is not directly involved in extracellular electron transfer, understanding its role in the context of G. sulfurreducens metabolism provides insights into bioremediation applications:

• Isoprenoid Production for Electron Transfer Components:

  • DXR catalyzes a critical step in the MEP pathway for isoprenoid biosynthesis

  • Isoprenoids are essential for the synthesis of menaquinones and ubiquinones, which are critical electron carriers in respiratory chains

  • G. sulfurreducens likely uses a menaquinol oxidase for oxygen reduction during microaerobic growth

• Integration with Metal Reduction Pathways:

  • G. sulfurreducens is known for its ability to reduce metals like uranium via extracellular electron transfer mechanisms

  • The cytochrome-rich outer membrane of G. sulfurreducens facilitates electron transfer to external acceptors

  • Recent research has revealed that microbial nanowires in G. sulfurreducens consist of the hexaheme cytochrome OmcS rather than PilA proteins as previously thought

• Metabolic Adaptation During Bioremediation:

  • During uranium bioremediation, G. sulfurreducens must adapt its metabolism to different electron acceptors

  • DXR activity may be modulated as part of metabolic adjustments during shifts between different electron acceptors

  • The MEP pathway likely plays a role in cell membrane adaptation when cells are exposed to contaminants

When studying G. sulfurreducens in bioremediation contexts, researchers should note that syntrophic growth with denitrifying microbial communities can accelerate denitrification and enhance removal of contaminants like nitrate , suggesting complex metabolic interactions that may involve isoprenoid-dependent processes.

What is known about the transcriptional regulation of DXR in Geobacter sulfurreducens under different environmental conditions?

The transcriptional regulation of DXR in G. sulfurreducens displays significant environmental responsiveness:

• Oxygen Exposure Response:

  • Under microaerobic conditions, G. sulfurreducens can utilize oxygen as a terminal electron acceptor

  • Transcriptome analysis suggests different survival strategies depending on oxygen concentration:

    • At low oxygen levels: motility genes upregulated to escape microaerobic areas

    • At moderate oxygen levels: genes for oxygen reduction coupled to cell growth

    • At high oxygen levels: genes for protective layer formation

  • DXR expression is likely modulated as part of these adaptive responses

• Electron Acceptor Influence:

  • Shifts in cytochrome expression occur depending on the electron acceptor used

  • When growing with different electron acceptors (fumarate vs. Fe(III) oxide), G. sulfurreducens modifies its surface chemistry, including lipopolysaccharide (LPS) structure

  • These adaptations suggest coordinated regulation of multiple metabolic pathways, potentially including the MEP pathway

• Growth Phase Dependencies:

  • In situ growth rates of G. sulfurreducens can be monitored through expression of ribosomal proteins like rpsC

  • Metabolic enzyme expression, including DXR, likely correlates with these growth rate indicators

  • Doubling times ranging from 6.56 h to 89.28 h have been observed under different conditions

How does Geobacter sulfurreducens DXR compare structurally and functionally to DXR enzymes from other organisms?

Comparative analysis reveals both conservation and divergence:

Key comparative insights:

• Structural Conservation:

  • DXR enzymes typically function as homodimers

  • Three-domain architecture with N-terminal cofactor binding, central catalytic, and C-terminal domains

  • NADPH binding region is highly conserved

  • Metal-binding sites coordinated by conserved acidic residues

• Functional Divergence:

  • Substrate affinity varies considerably between species

  • Inhibitor sensitivity shows species-specific patterns

  • Optimal pH and temperature conditions reflect the organism's ecological niche

• Evolutionary Considerations:

  • The MEP pathway is absent in mammals but present in many bacteria and some eukaryotes

  • The pathway's presence in G. sulfurreducens reflects its bacterial lineage

  • Horizontal gene transfer may have contributed to MEP pathway distribution across taxa

This comparative understanding is essential when developing inhibitors or considering the enzyme's role in different metabolic contexts across species.

What approaches can be used to study DXR inhibition in Geobacter sulfurreducens and how might this inform antimicrobial development?

Several strategic approaches can be employed:

• Structure-Based Drug Design Approaches:

  • In the absence of a crystal structure for G. sulfurreducens DXR, homology modeling based on related DXR structures can guide inhibitor design

  • Molecular docking simulations can predict binding modes and affinities

  • Fragment-based screening can identify new scaffolds for inhibitor development

• Known DXR Inhibitor Adaptation:

  • Fosmidomycin, the best-characterized DXR inhibitor, and its derivatives can be tested against G. sulfurreducens DXR

  • Species-specific differences in fosmidomycin sensitivity indicate the need for customized inhibitor design

  • Lipophilic modifications to increase cell penetration, as shown effective for M. tuberculosis DXR

• Growth Inhibition Studies:

  • Establishing minimal inhibitory concentration (MIC) values for DXR inhibitors against G. sulfurreducens

  • Correlation between enzymatic inhibition (IC₅₀) and growth inhibition (MIC)

  • Assessment of inhibitor effects under different growth conditions (anaerobic vs. microaerobic)

• Translational Relevance to Antimicrobial Development:

  • The MEP pathway's absence in humans makes it an attractive target for selective antimicrobial development

  • G. sulfurreducens research can inform strategies for targeting related deltaproteobacteria

  • Environmental considerations for antimicrobials targeting beneficial environmental bacteria like Geobacter species

When designing inhibitor studies, it's important to consider that G. sulfurreducens can survive toxicity challenges, as demonstrated in studies with copper where it can form Cu₂S nanoparticles via bioreduction . This reflects the organism's versatile detoxification mechanisms that might influence drug efficacy.

What simulation tools and computational approaches are most effective for studying Geobacter sulfurreducens DXR structure-function relationships?

A multi-layered computational approach is recommended:

• Homology Modeling:

  • Software: SWISS-MODEL, Phyre2, Modeller

  • Template selection: Crystal structures of DXR from related bacteria (E. coli, M. tuberculosis)

  • Quality assessment: PROCHECK, QMEAN, MolProbity

  • Model refinement: Energy minimization using molecular dynamics

• Molecular Dynamics Simulations:

  • Software packages: GROMACS, AMBER, NAMD

  • Simulation conditions: Include NADPH, Mg²⁺, and water molecules in the simulation box

  • Analysis of protein flexibility and conformational changes upon substrate binding

  • Typical simulation times: 100-500 ns for adequate sampling

• Substrate and Inhibitor Docking:

  • Software: AutoDock Vina, GOLD, Glide

  • Preparation of ligand libraries: DXP analogs, known DXR inhibitors

  • Scoring functions: Combination of force field-based and empirical scoring

  • Virtual screening approaches for identifying novel inhibitors

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Essential for studying the reaction mechanism

  • QM region: Active site residues, substrate, cofactor

  • MM region: Remainder of the protein and solvent

  • Software: Gaussian/AMBER interface, ONIOM

These computational approaches should be validated experimentally using site-directed mutagenesis of predicted key residues followed by kinetic analyses.

How can advanced imaging techniques be applied to study the localization and interaction partners of DXR in Geobacter sulfurreducens?

Several cutting-edge imaging approaches can provide valuable insights:

• Super-Resolution Microscopy:

  • STORM or PALM microscopy with fluorescently tagged DXR can reveal subcellular localization with ~20 nm resolution

  • Multicolor imaging allows co-localization studies with other enzymes in the MEP pathway

  • Sample preparation: Fixation methods must preserve the anaerobic state of G. sulfurreducens

• Cryo-Electron Microscopy:

  • Single-particle cryo-EM can resolve protein structure at near-atomic resolution

  • Cryo-electron tomography can visualize DXR in its cellular context

  • Immunogold labeling can identify DXR within tomograms

• Protein-Protein Interaction Mapping:

  • Proximity labeling approaches (BioID, APEX) can identify proteins in close proximity to DXR in vivo

  • Co-immunoprecipitation followed by mass spectrometry can identify stable interaction partners

  • Fluorescence resonance energy transfer (FRET) can confirm direct interactions with candidate partners

• Correlative Light and Electron Microscopy (CLEM):

  • Combines the specificity of fluorescence microscopy with the resolution of electron microscopy

  • Particularly valuable for studying membrane-associated processes in G. sulfurreducens

  • Can reveal DXR localization in relation to cell ultrastructure

When applying these techniques to G. sulfurreducens, special consideration should be given to maintaining anaerobic conditions during sample preparation, as exposure to oxygen can alter protein localization and cellular morphology.

How might the DXR pathway in Geobacter sulfurreducens be engineered for enhanced bioremediation capabilities?

Several promising engineering strategies can be pursued:

• Metabolic Flux Enhancement:

  • Overexpression of DXR to increase flux through the MEP pathway

  • Fine-tuning expression of upstream DXS and downstream MEP pathway enzymes to prevent bottlenecks

  • Directed evolution of DXR for improved catalytic efficiency under bioremediation conditions

• Engineered Syntrophic Relationships:

  • Co-culture systems with denitrifying bacteria to enhance nitrate bioremediation

  • Engineering G. sulfurreducens to enhance the demonstrated syntrophic growth effect that accelerates denitrification by 13-51%

  • Development of artificial consortia with optimized electron transfer between species

• Environmental Adaptation Enhancements:

  • Engineering stress response systems linked to isoprenoid biosynthesis

  • Modification of cell surface structures controlled by MEP pathway products

  • Enhancing resistance to contaminants like uranium by optimizing metabolic responses

• Integration with Bioelectrochemical Systems:

  • Optimization of DXR and the MEP pathway to support enhanced electron transfer in microbial fuel cells

  • Engineering strains with improved electroactive biofilm formation capabilities

  • Developing strains with reduced startup time for bioelectrochemical systems

The effectiveness of these approaches can be evaluated using pre-pilot scale systems like those described for microbial electrochemistry testing , where different G. sulfurreducens inocula with varying physiologies showed significant differences in electroactive response.

What are the most promising research questions regarding post-translational modifications and regulation of DXR in Geobacter sulfurreducens?

Several intriguing research directions warrant investigation:

• Phosphorylation Dynamics:

  • Identification of kinases/phosphatases that regulate DXR activity

  • Mapping of phosphorylation sites using phosphoproteomics

  • Functional consequences of phosphorylation on enzyme kinetics and stability

  • Research question: Does phosphorylation serve as a rapid response mechanism to changing environmental conditions?

• Redox Regulation:

  • Impact of cellular redox state on DXR activity

  • Identification of critical cysteine residues susceptible to oxidation

  • Connection to the microaerobic growth capabilities of G. sulfurreducens

  • Research question: How does G. sulfurreducens protect DXR from oxidative damage during oxygen exposure?

• Protein-Protein Interactions:

  • Identification of DXR interaction partners beyond the MEP pathway

  • Potential formation of metabolons (enzyme complexes) for metabolic channeling

  • Interaction with membrane components and cytochromes

  • Research question: Does DXR form part of a larger metabolic complex that regulates isoprenoid biosynthesis?

• Membrane Association Dynamics:

  • Conditions triggering membrane association/dissociation

  • Lipid interactions influencing enzyme activity

  • Connection to the rough lipopolysaccharide synthesis adapted for different electron acceptors

  • Research question: How does membrane composition affect DXR localization and activity?