Recombinant Photobacterium profundum 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is Photobacterium profundum dxr and why is it significant for research?

Photobacterium profundum dxr (1-deoxy-D-xylulose 5-phosphate reductoisomerase) is the second enzyme in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. This enzyme catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP) through an isomerization followed by an NADPH-dependent reduction .

P. profundum dxr is particularly significant because it comes from a deep-sea bacterium that has adapted to high-pressure environments. P. profundum strain SS9 was isolated from the Sulu Trough and represents a barophilic (pressure-loving) organism that can grow at pressures between 0.1 MPa and 70 MPa, with optimal growth at 10 MPa . This makes its enzymes, including dxr, valuable models for studying pressure adaptation of proteins.

The MEP pathway is absent in humans but present in many bacteria, algae, plants, and the malarial parasite Plasmodium falciparum, making dxr an attractive target for the development of new antimalarial and antibacterial compounds .

How is recombinant P. profundum dxr typically expressed and purified?

Methodology for expressing and purifying recombinant P. profundum dxr:

• Expression System: The recombinant protein is typically expressed in E. coli expression systems .

• Expression Vector Construction:

  • The full-length dxr gene (encoding amino acids 1-397) is amplified from P. profundum SS9 genomic DNA

  • The gene is inserted into an appropriate expression vector containing an inducible promoter

  • A His-tag or other affinity tag may be added to facilitate purification

• Culture Conditions:

  • Transform the expression vector into an E. coli strain optimized for protein expression

  • Grow cultures at lower temperatures (15-25°C) to enhance proper folding

  • Induce expression when cultures reach mid-log phase

• Purification Protocol:

  • Harvest cells and lyse using appropriate buffer systems

  • Perform initial purification using affinity chromatography

  • Further purify using ion exchange and/or size exclusion chromatography

  • The final purity of commercially available recombinant P. profundum dxr is typically >85% as determined by SDS-PAGE

• Storage:

  • For long-term storage, the purified protein can be stored at -20°C/-80°C with 5-50% glycerol

  • The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C

  • Lyophilized forms can be stored for up to 12 months at -20°C/-80°C

How does the MEP pathway in P. profundum compare to other bacterial systems?

The MEP pathway in P. profundum shares fundamental similarities with other bacterial systems but with notable adaptations for deep-sea environments:

• Pathway Conservation: Like other bacteria, P. profundum utilizes the complete MEP pathway starting with the condensation of pyruvate and glyceraldehyde-3-phosphate by DXS (1-deoxy-D-xylulose 5-phosphate synthase), followed by the conversion of DXP to MEP by dxr .

• Expression Regulation: In P. profundum, ToxR, a transmembrane DNA-binding protein, plays a role in the regulation of genes in a pressure-dependent manner . While specific regulation of dxr by ToxR hasn't been directly demonstrated, the transcriptional landscape of P. profundum shows unique adaptations to high-pressure environments that likely affect MEP pathway enzymes .

• Pressure Adaptation: P. profundum is a moderately barophilic bacterium capable of growth at pressures between 0.1 MPa and 70 MPa . This suggests that its MEP pathway enzymes, including dxr, have evolved structural adaptations that maintain functionality under high hydrostatic pressure.

• Genomic Context: RNA-seq analysis of P. profundum has revealed a complex transcriptional landscape and potential regulatory networks that may influence the expression of MEP pathway genes under different pressure conditions .

What are the optimal conditions for assaying P. profundum dxr enzymatic activity?

Based on the characteristics of P. profundum and general properties of dxr enzymes, the following protocol is recommended for optimal assay conditions:

Recommended Assay Protocol:

• Buffer Composition:

  • 100 mM Tris-HCl or HEPES buffer (pH 7.5-8.0)

  • 1-5 mM MgCl₂ (essential cofactor)

  • 0.1-0.5 mM NADPH (electron donor)

  • 0.1-1.0 mM DXP (substrate)

• Temperature Conditions:

  • Primary assay at 10-15°C (matching P. profundum's optimal growth temperature)

  • Comparative assays at 4°C and 25°C to assess temperature dependence

• Pressure Conditions:

  • Standard atmospheric pressure (0.1 MPa) for baseline activity

  • High-pressure assays at 10 MPa (optimal growth pressure) and 28 MPa using specialized high-pressure equipment

• Activity Measurement Methods:

  • Spectrophotometric monitoring of NADPH oxidation at 340 nm

  • HPLC analysis of DXP consumption and MEP formation

  • Mass spectrometry for precise quantification of reaction products

• Controls and Validation:

  • Include fosmidomycin (known dxr inhibitor) as a negative control

  • Run parallel assays with E. coli dxr for comparison

  • Verify the identity of reaction products using LC-MS/MS

How do pressure and temperature affect the stability and activity of P. profundum dxr?

P. profundum is adapted to the deep-sea environment, with optimal growth at 10°C and 10 MPa pressure . These environmental adaptations are reflected in the properties of its enzymes, including dxr:

Pressure Effects:

• P. profundum shows growth capability at pressures between 0.1 MPa and 70 MPa, with optimal growth at 10 MPa

• RNA-seq analysis of P. profundum under different pressure conditions (0.1 MPa vs. 28 MPa) reveals significant changes in gene expression

• While specific data on dxr pressure response is not directly provided, proteins from piezophilic organisms typically maintain structural integrity and function at elevated pressures where mesophilic homologs would denature

Temperature Effects:

• P. profundum is capable of growth between 4°C and 18°C at atmospheric pressure, with optimal growth at 10°C

• As a psychrophilic/psychrotolerant organism, its enzymes, including dxr, are expected to show:

  • Higher catalytic efficiency at low temperatures

  • Greater structural flexibility

  • Lower thermal stability compared to mesophilic homologs

Combined Effects:
The adaptation to both cold and high-pressure environments likely results in unique structural features in P. profundum dxr that balance flexibility needed for low-temperature activity with stability required under high pressure.

What are potential applications of recombinant P. profundum dxr in biocatalysis and high-pressure biotechnology?

Recombinant P. profundum dxr offers several promising applications in biocatalysis and high-pressure biotechnology due to its unique adaptations:

Biocatalytic Applications:

• Terpenoid Biosynthesis: The MEP pathway is crucial for terpenoid production. Studies with other DXS and DXR enzymes show that overexpression can significantly increase diterpene yields in transgenic systems . P. profundum dxr could be exploited for:

  • Production of rare marine isoprenoids

  • Enhanced synthesis of pharmacologically active terpenoids

  • Development of cold-active isoprenoid production systems

• Cold-Active Biocatalysis: As an enzyme from a psychrophilic organism, P. profundum dxr likely exhibits:

  • Higher catalytic activity at low temperatures

  • Reduced energy requirements for industrial processes

  • Compatibility with heat-sensitive substrates or products

High-Pressure Biotechnology Applications:

• Model System for Pressure Effects: P. profundum dxr can serve as a model to understand:

  • Molecular mechanisms of enzyme adaptation to high pressure

  • Structure-function relationships under extreme conditions

  • Evolutionary strategies for pressure adaptation

• Pressure-Enhanced Biosynthesis:

  • Development of high-pressure bioreactors for isoprenoid production

  • Exploration of novel reaction conditions that may enhance selectivity or yield

  • Investigation of pressure effects on reaction equilibria and rates

• Protein Engineering Platform:

  • Identification of pressure-stabilizing motifs that could be transferred to other enzymes

  • Development of enzymes functioning in deep-sea environments for in situ bioremediation

  • Creation of pressure-resistant biocatalysts for industrial applications

How might genetic manipulation of the dxr gene in P. profundum affect its pressure adaptation?

Genetic manipulation of the dxr gene in P. profundum could provide valuable insights into pressure adaptation mechanisms:

Potential Experimental Approaches:

• Gene Knockout/Knockdown Studies:

  • Creation of dxr deletion mutants similar to methodologies used for flaA deletion in P. profundum

  • Complementation with dxr genes from different pressure environments

  • Assessment of growth under various pressure conditions

• Site-Directed Mutagenesis:

  • Targeted modification of residues predicted to be involved in pressure adaptation

  • Creation of chimeric enzymes combining domains from piezophilic and non-piezophilic homologs

  • Introduction of stabilizing/destabilizing mutations to probe pressure effects

• Expression Analysis Under Varying Pressure:

  • Similar to studies with ToxR-regulated genes , examine dxr expression under different pressure conditions

  • Identify pressure-responsive regulatory elements in the dxr promoter region

  • Assess the role of ToxR in dxr regulation under high pressure

Expected Outcomes and Interpretations:

• Growth Phenotypes:

  • Mutations disrupting pressure-adaptive features might restrict growth at high pressures

  • Compensatory mutations in other MEP pathway enzymes could arise

  • Changes in isoprenoid composition might occur to maintain membrane integrity

• Enzymatic Activity Profiles:

  • Modified pressure optima for mutant enzymes

  • Altered kinetic parameters (Km, kcat) as a function of pressure

  • Potential trade-offs between pressure resistance and catalytic efficiency

• Cellular Response:

  • Transcriptional changes in related metabolic pathways

  • Modifications in membrane composition to compensate for altered isoprenoid biosynthesis

  • Potential activation of stress response mechanisms

What techniques are most effective for studying P. profundum dxr under high-pressure conditions?

Studying enzymes under high-pressure conditions presents unique challenges and requires specialized techniques:

High-Pressure Enzyme Assay Methods:

• Stopped-Flow High-Pressure Spectroscopy:

  • Allows measurement of reaction kinetics under pressure

  • Can monitor NADPH oxidation at 340 nm to follow dxr activity

  • Provides real-time data on pressure effects on reaction rates

• High-Pressure Bioreactors:

  • Enable longer-term reactions under controlled pressure

  • Allow sampling for product analysis via HPLC or MS

  • Can be coupled with in-line analytical techniques

• Pressure Perturbation Calorimetry:

  • Measures volumetric changes associated with enzyme-substrate interactions

  • Provides thermodynamic parameters under pressure

  • Helps understand mechanistic details of pressure effects

Structural Analysis Under Pressure:

• High-Pressure X-ray Crystallography:

  • Visualizes structural changes in the enzyme under pressure

  • Requires specialized pressure cells for crystal mounting

  • Can reveal pressure-induced conformational changes

• High-Pressure NMR Spectroscopy:

  • Monitors protein dynamics and ligand interactions under pressure

  • Provides atomic-level insights into pressure effects

  • Allows titration of pressure to identify transition points

• Small-Angle X-ray Scattering (SAXS):

  • Examines global protein shape and oligomeric state under pressure

  • Works with proteins in solution

  • Can detect pressure-induced dissociation or association

Computational Approaches:

• Molecular Dynamics Simulations:

  • Model enzyme behavior under different pressure conditions

  • Predict pressure-sensitive regions and conformational changes

  • Test hypotheses about pressure adaptation mechanisms

• Comparative Genomics and Structural Bioinformatics:

  • Identify conserved features in pressure-adapted enzymes

  • Predict key residues involved in pressure adaptation

  • Guide experimental design for mutagenesis studies

How can structural information about P. profundum dxr contribute to the design of novel antimicrobial compounds?

The methylerythritol phosphate (MEP) pathway is absent in humans but present in many bacteria and the malaria parasite, making dxr an attractive target for antimicrobial and antimalarial drug development :

Drug Design Strategies Based on P. profundum dxr:

• Structure-Based Drug Design:

  • Crystal structures of P. profundum dxr in complex with inhibitors could reveal unique binding pocket features

  • Pressure-adapted enzymes often have distinct conformational flexibility that may offer novel inhibitor binding modes

  • Comparative analysis with pathogen dxr structures could identify selective targeting opportunities

• Exploitation of Known Inhibitors:

  • Fosmidomycin is a known inhibitor of dxr and shows promise as an antimalarial drug

  • Testing fosmidomycin analogs against P. profundum dxr could provide insights into structure-activity relationships

  • The following table summarizes potential comparative inhibition studies:

• Insights from Pressure Adaptation:

  • Pressure-adapted enzymes often exhibit altered active site architecture and dynamics

  • Features that maintain activity under pressure might reveal new druggable sites

  • Understanding how substrate binding is maintained under pressure could inform inhibitor design

Advantages of Using P. profundum dxr in Drug Discovery:

• Evolutionary Insights:

  • As a deep-sea bacterium, P. profundum represents a distinct evolutionary adaptation

  • Comparison with pathogen dxr enzymes may reveal conserved features essential for function versus adaptive variations

  • This could help design broad-spectrum inhibitors targeting essential, conserved features

• Biophysical Properties:

  • Pressure-adapted enzymes often display distinct conformational ensembles

  • Studies of P. profundum dxr under varying pressures could reveal conformational states not observed in standard conditions

  • These alternative conformations might offer novel binding pockets for inhibitor design

• Resistance Mechanisms:

  • Understanding natural variations in dxr across different species could predict potential resistance mechanisms

  • P. profundum dxr may possess structural features that could inform design of inhibitors less prone to resistance development