Recombinant Photobacterium profundum 1-deoxy-D-xylulose-5-phosphate synthase (dxs), partial

What is 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and what role does it play in bacterial metabolism?

1-deoxy-D-xylulose-5-phosphate synthase (DXS) is a thiamine pyrophosphate-dependent enzyme that catalyzes the first and rate-limiting step in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. DXS catalyzes the acyloin condensation reaction between carbon atoms 2 and 3 of pyruvate and D-glyceraldehyde 3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (DXP) .

This reaction is particularly significant because DXP serves as a precursor not only for isoprenoids but also for thiamine (vitamin B1) and pyridoxol (vitamin B6) biosynthesis. The MEP pathway is the exclusive route for isoprenoid synthesis in most bacteria, algae, and plant plastids, contrasting with the mevalonate pathway used by archaea, fungi, and animals .

The structural and functional significance of DXS makes it an attractive target for the development of novel antibiotics, antimalarials, and herbicides. Understanding its function in pressure-adapted organisms like P. profundum can provide additional insights into enzymatic adaptation mechanisms.

What are the unique features of Photobacterium profundum that make its DXS of interest to researchers?

Photobacterium profundum is a deep-sea Gammaproteobacterium with remarkable adaptability to extreme environmental conditions. Several features make its DXS enzyme particularly interesting for research:

• Psychrophilic and piezophilic adaptation: P. profundum strain SS9 grows optimally at 15°C and 28 MPa, making it both a psychrophile (cold-loving) and a piezophile (pressure-loving) organism .

• Model organism status: Despite its preference for high-pressure environments, P. profundum can grow at atmospheric pressure, facilitating genetic manipulation and laboratory culture, which has established it as a model organism for studying pressure adaptation .

• Experimental versatility: The ability to cultivate P. profundum under varying pressure conditions (from 0.1 MPa to 70 MPa) allows for comparative studies of protein expression and enzyme activity .

• Metabolic adaptability: Proteomic studies have shown that proteins involved in the glycolysis/gluconeogenesis pathway are up-regulated at high pressure, while those in oxidative phosphorylation are up-regulated at atmospheric pressure, suggesting complex metabolic adjustments to pressure changes .

These characteristics indicate that P. profundum DXS may possess unique structural and catalytic properties that enable it to function efficiently under conditions that would typically impair enzyme activity. This makes it valuable for studying fundamental principles of enzyme adaptation to extreme environments.

How is recombinant P. profundum DXS typically expressed in laboratory settings?

While the search results don't provide specific protocols for P. profundum DXS expression, we can outline a general methodology based on approaches used for similar enzymes and other P. profundum proteins:

• Gene amplification and cloning:

  • PCR amplification of the dxs gene from P. profundum genomic DNA

  • Cloning into an appropriate expression vector with a selectable marker and inducible promoter

  • Addition of an affinity tag (commonly His₆-tag) for purification purposes

• Expression host selection:

  • E. coli is the most common expression host, with BL21(DE3) or its derivatives frequently used

  • The choice between E. coli and other expression systems should consider codon usage, post-translational modifications, and solubility requirements

• Culture conditions:

  • Culture temperature typically reduced to 15-20°C after induction to enhance soluble protein expression

  • Extended induction times (overnight to 24 hours) at lower temperatures

  • Use of rich media supplemented with appropriate antibiotics

• Protein extraction and purification:

  • Cell lysis by sonication or French press in a buffer containing protease inhibitors

  • Clarification of lysate by centrifugation

  • Affinity chromatography using Ni-NTA resin for His-tagged proteins

  • Further purification by ion exchange or size exclusion chromatography

• Activity preservation:

  • Addition of thiamine pyrophosphate (TPP) and divalent cations (like Mg²⁺) in purification buffers

  • Storage in glycerol-containing buffers at -80°C for long-term preservation

Similar approaches have been successfully used for DXS from Rhodobacter capsulatus, where the enzyme was purified to >95% homogeneity in two steps and appeared to be a homodimer with 68 kDa subunits .

What are the structural domains of DXS and their functions?

Based on crystallographic studies of DXS from other bacteria, the enzyme typically contains three distinct domains with specific functions:

Each of these domains bears homology to equivalent domains in related thiamine diphosphate-dependent enzymes, such as transketolase and the E1 subunit of pyruvate dehydrogenase .

A distinctive feature of DXS compared to related TPP-dependent enzymes is its domain arrangement. Unlike other TPP-dependent enzymes where the active site is situated at a chain interface, in DXS, the active site resides between two domain faces within the same monomer of a homodimeric complex .

This unique structural arrangement may contribute to the specific catalytic properties of DXS and its ability to catalyze the condensation of pyruvate and D-glyceraldehyde 3-phosphate, rather than the decarboxylation reactions typically catalyzed by other TPP-dependent enzymes.

What cofactors are required for DXS enzymatic activity?

DXS requires specific cofactors for its catalytic activity:

• Thiamine pyrophosphate (TPP): The primary cofactor essential for the catalytic mechanism. TPP forms a covalent adduct with pyruvate, generating an activated intermediate for the condensation reaction .

• Divalent metal ions: Typically Mg²⁺ or other divalent cations that help coordinate the pyrophosphate moiety of TPP in the active site.

• Substrate requirements: The enzymatic reaction requires:

  • Pyruvate

  • D-glyceraldehyde 3-phosphate (GAP)

The reaction proceeds through multiple steps:

• TPP activates pyruvate through nucleophilic attack

• Decarboxylation generates an activated TPP-bound intermediate

• The intermediate reacts with GAP

• The condensation product 1-deoxy-D-xylulose-5-phosphate (DXP) is released, regenerating the free enzyme

Assays for DXS activity typically include TPP, Mg²⁺, pyruvate, and GAP, with activity monitored through either the consumption of substrates or the formation of DXP.

How do pressure and temperature affect the kinetic parameters of P. profundum DXS?

While specific data for P. profundum DXS are not provided in the search results, insights can be drawn from studies on other P. profundum enzymes, such as α-carbonic anhydrase (PprCA):

PprCA exhibits maximal catalytic activity at psychrophilic temperatures with substantial decreases in activity at mesophilic and thermophilic ranges. It also demonstrates salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions .

For P. profundum DXS, similar adaptations might be expected:

• Pressure effects on enzyme kinetics:

  • Increased catalytic efficiency (kcat/Km) at higher pressures (around 28 MPa)

  • Potential alterations in substrate binding affinity (Km) as a function of pressure

  • Modified reaction mechanism or rate-limiting step under pressure

• Temperature adaptation:

  • Higher activity at lower temperatures (10-15°C) compared to mesophilic homologs

  • Reduced thermal stability but enhanced catalytic efficiency at low temperatures

  • Potential cold-adaptation features: increased structural flexibility, reduced number of stabilizing interactions, and modified surface charge distribution

• Combined pressure and temperature effects:

  • Synergistic effects where optimal temperature may shift with pressure changes

  • Possible alterations in activation energy and thermodynamic parameters

To study these effects, researchers would need to:

• Design high-pressure enzymatic assays using pressure vessels

• Measure reaction rates at various pressure and temperature combinations

• Determine kinetic parameters (Km, Vmax, kcat) under different conditions

• Analyze thermodynamic parameters (ΔH, ΔS, ΔG) as a function of pressure

Such studies would provide valuable insights into the molecular adaptations that enable enzymes to function efficiently in deep-sea environments.

What are the best methods for assaying DXS activity in vitro?

Several approaches can be employed to measure DXS activity in vitro, each with specific advantages:

• Direct measurement of DXP formation:

  • High-performance liquid chromatography (HPLC) with detection of DXP

  • Liquid chromatography-mass spectrometry (LC-MS) for higher sensitivity and specificity

  • For P. profundum experiments, methods similar to those used for piscibactin detection could be adapted, using HLB cartridges and HPLC separation

• Coupled enzyme assays:

  • Using 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) as a coupling enzyme

  • Monitoring NADPH oxidation spectrophotometrically at 340 nm

  • This approach provides a continuous readout of DXS activity

• Radiometric assays:

  • Using ¹⁴C-labeled pyruvate as a substrate

  • Measuring incorporation of radioactivity into DXP

  • Separation of unreacted substrate from product by ion-exchange chromatography

• Specialized assay for pressure studies:

  • Design of pressure vessels containing reaction mixtures

  • Sampling and analysis after pressure treatment

  • Use of stopped-flow techniques adapted for high pressure

A new assay developed for 1-deoxy-D-xylulose 5-phosphate synthase A and B from Rhodobacter capsulatus determined the following steady-state kinetic constants:

• K(m)(pyruvate) = 0.61 and 3.0 mM

• K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 μM

• V(max) = 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4)

Similar approaches could be adapted for P. profundum DXS, with appropriate modifications to account for the enzyme's adaptation to cold and high-pressure environments.

How does the structure of P. profundum DXS compare to DXS from mesophilic organisms?

Although specific structural data for P. profundum DXS is not provided in the search results, we can predict potential structural adaptations based on:

• Known DXS structures from mesophilic bacteria like E. coli and Deinococcus radiodurans

• Structural adaptations observed in other psychrophilic and piezophilic enzymes, including PprCA from P. profundum

Expected structural differences may include:

• Cold adaptation features:

  • Reduced number of stabilizing interactions (fewer salt bridges, hydrogen bonds)

  • Increased surface hydrophilicity

  • Greater flexibility in catalytic loops and domains

  • More accessible active site

  • Modified electrostatic potential on protein surface

• Pressure adaptation features:

  • Altered oligomeric state or interface interactions

  • Modified cavity distribution to minimize volume changes during catalysis

  • Increased ion pairs or hydrophobic interactions in specific regions

  • Potential chloride ion binding sites, as observed in PprCA

• Domain-specific adaptations:

  • Modifications in domain interfaces to maintain flexibility under pressure

  • Altered substrate binding pocket architecture

  • Modified cofactor binding regions to maintain optimal binding under pressure

The crystal structure of PprCA from P. profundum revealed a unique chloride ion in the dimer interface, which has not been observed in any other α-CAs characterized to date . Similar novel structural features might be present in P. profundum DXS, potentially contributing to its adaptation to the deep-sea environment.

To fully understand these adaptations, X-ray crystallography or cryo-electron microscopy studies of P. profundum DXS under varying pressure conditions would be necessary, complemented by molecular dynamics simulations to analyze pressure effects on protein dynamics.

What experimental approaches can be used to study the pressure adaptation mechanisms of P. profundum DXS?

Investigating pressure adaptation mechanisms in P. profundum DXS requires specialized techniques:

• High-pressure enzyme kinetics:

  • Measurement of enzymatic activity at varying pressures (0.1-70 MPa)

  • Determination of pressure-dependent kinetic parameters

  • Analysis of activation volume (ΔV‡) through pressure-dependent rate constants

• Structural biology under pressure:

  • High-pressure X-ray crystallography

  • High-pressure NMR spectroscopy

  • Small-angle X-ray scattering (SAXS) under pressure to analyze conformational changes

• Molecular dynamics simulations:

  • Simulation of protein behavior under various pressure conditions

  • Analysis of pressure effects on protein flexibility, hydration, and substrate binding

  • Similar to chloride ion occupancy analysis performed for PprCA, which showed 100% occupancy for the Cl⁻ ion in the dimer interface

• Comparative studies:

  • Expression of DXS from P. profundum and mesophilic counterparts

  • Comparison of stability and activity under varying pressure conditions

  • Chimeric enzyme construction to identify pressure-sensing domains

• Mutagenesis approaches:

  • Site-directed mutagenesis targeting residues predicted to be involved in pressure adaptation

  • Creation of mutants with altered pressure sensitivity

  • Domain swapping between pressure-adapted and mesophilic DXS

• Proteomic analysis:

  • Label-free quantitative proteomic analysis similar to that used for studying P. profundum under different pressure regimes

  • Comparison of post-translational modifications in response to pressure changes

• Growth and expression studies:

  • Cultivation of P. profundum under different pressure conditions using specialized equipment

  • Analysis of DXS expression levels and modifications

  • Methods similar to those used for P. profundum culture in pressure vessels at 28 MPa

These approaches would provide complementary insights into the molecular mechanisms that enable P. profundum DXS to function efficiently under high-pressure conditions of the deep sea.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of P. profundum DXS?

Site-directed mutagenesis represents a powerful approach to probe the catalytic mechanism and pressure adaptation of P. profundum DXS. The following strategy could be implemented:

• Identification of target residues:

  • Catalytic residues involved in TPP binding and activation

  • Substrate binding pocket residues interacting with pyruvate and GAP

  • Interface residues potentially involved in pressure sensing

  • Residues unique to P. profundum DXS compared to mesophilic homologs

• Designing mutation strategies:

  • Conservative mutations to assess the importance of specific chemical properties

  • Introduction of residues found in mesophilic homologs to test pressure adaptation

  • Alanine-scanning mutagenesis of selected regions

  • Introduction or removal of potential salt bridges and hydrogen bonds

• Mutation protocol:

  • PCR-based site-directed mutagenesis similar to construction of kanamycin cassette-marked versions used in P. damselae

  • Use of overlap extension PCR to create mutations

  • Construction of expression vectors containing mutated genes

• Functional analysis of mutants:

  • Expression and purification of mutant proteins

  • Kinetic characterization under varying pressure and temperature conditions

  • Stability assessment through thermal and pressure denaturation studies

  • Structural analysis of selected mutants

• Specific targets for investigation:

  • TPP-binding residues in Domain I

  • Residues involved in the coordination of the divalent metal ion

  • Active site residues participating in substrate binding and catalysis

  • Flexible regions that might function as pressure sensors

By systematically analyzing the effects of mutations on enzyme activity, stability, and pressure response, researchers can delineate the structural features responsible for the unique properties of P. profundum DXS and potentially engineer enzymes with enhanced pressure tolerance for biotechnological applications.

What are the challenges in expressing and purifying active recombinant DXS from P. profundum?

Expressing and purifying active recombinant DXS from P. profundum presents several unique challenges:

• Codon usage bias:

  • P. profundum has a different codon usage pattern compared to common expression hosts

  • Potential solution: Codon optimization of the dxs gene for the expression host or use of host strains supplying rare tRNAs

• Protein solubility and folding:

  • DXS is a complex multi-domain enzyme that may fold incorrectly under standard expression conditions

  • Strategies to address this include:

    • Reduced expression temperature (15-18°C)

    • Co-expression with chaperones (DnaK, DnaJ, GroEL/GroES) that are naturally up-regulated in P. profundum

    • Use of fusion partners to enhance solubility

• Pressure and temperature adaptation:

  • The enzyme is naturally adapted to function at high pressure (28 MPa) and low temperature (15°C)

  • Expression at atmospheric pressure may affect folding and activity

  • Consideration of post-expression treatments under pressure may be necessary

• Cofactor requirements:

  • Ensuring proper incorporation of TPP and divalent cations during purification

  • Addition of cofactors to stabilize the enzyme during purification

• Protein stability:

  • Psychrophilic enzymes often show reduced thermal stability

  • Rapid handling at low temperatures is required

  • Use of appropriate stabilizers and protease inhibitors

• Activity preservation:

  • Cold-adapted enzymes may lose activity rapidly

  • Storage conditions must be carefully optimized

  • Consider flash-freezing in small aliquots with cryoprotectants

• Expression systems:

  • While E. coli is commonly used, alternative systems might be beneficial

  • A system similar to that used for PprCA expression could be adapted

Successfully addressing these challenges requires careful optimization of expression conditions, purification protocols, and storage methods to preserve the unique properties of this deep-sea bacterial enzyme.

How can isotopic labeling be used to track DXS activity in metabolic studies?

Isotopic labeling represents a powerful approach for tracking DXS activity and studying the MEP pathway in P. profundum:

• Types of isotopic labeling for DXS studies:

  • ¹³C-labeled pyruvate or glucose as primary carbon sources

  • ²H-labeled substrates for studying hydrogen transfer steps

  • ¹⁵N-labeled precursors for studying nitrogen-containing products

• Experimental design for in vivo studies:

  • Growth of P. profundum with isotopically labeled substrates under different pressure conditions

  • Extraction and analysis of metabolites

  • Tracing carbon flow through the MEP pathway

  • Culture methods similar to those used for P. profundum in Pasteur pipettes under controlled pressure

• Detection and analysis methods:

  • Gas chromatography-mass spectrometry (GC-MS) for volatile compounds

  • Liquid chromatography-mass spectrometry (LC-MS) for non-volatile intermediates

  • Nuclear magnetic resonance (NMR) spectroscopy for structural determination

  • Similar approaches to those used for detecting compounds in P. damselae supernatants

• Specific applications:

  • Determining flux through the MEP pathway under different pressure conditions

  • Identifying bottlenecks in isoprenoid biosynthesis

  • Studying the effect of pressure on metabolic partitioning

  • Investigating crossover between MEP and other metabolic pathways

• Data analysis approaches:

  • Metabolic flux analysis using isotopomer distributions

  • Kinetic modeling of pathway dynamics

  • Partial least squares (PLS) approaches for complex datasets

• Combined approaches:

  • Integration of transcriptomic, proteomic, and metabolomic data

  • Use of isotope labeling to validate findings from other omics approaches

  • Design of fractional factorial experiments to optimize multiple parameters

These approaches would provide detailed insights into the function of DXS within the cellular context of P. profundum and how its activity is modulated by environmental factors like pressure and temperature.

What are the considerations for designing partial constructs of P. profundum DXS for domain-specific studies?

Designing partial constructs of P. profundum DXS requires careful consideration of domain boundaries, protein stability, and functional requirements:

• Domain boundary identification:

  • Bioinformatic analysis using:

    • Multiple sequence alignment with characterized DXS enzymes

    • Secondary structure prediction

    • Domain prediction algorithms

  • Structural homology modeling based on crystal structures of DXS from E. coli and D. radiodurans

  • Consideration of interdomain linker regions

• Design strategies for partial constructs:

  • Individual domain expression (Domains I, II, or III)

  • Combined domain constructs (Domains I-II or II-III)

  • Systematic truncation series

  • Introduction of stabilizing mutations at domain interfaces

• Expression optimization considerations:

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

  • Design of construct-specific purification strategies

  • Optimization of expression conditions for each construct

  • Selection of appropriate expression hosts

• Functional analysis approaches:

  • Cofactor binding studies for Domain I constructs

  • Substrate binding assays for Domain II constructs

  • Oligomerization analysis for constructs containing Domain III

  • Complementation studies between separate domains

• Structural characterization:

  • X-ray crystallography of individual domains

  • NMR spectroscopy for smaller domain constructs

  • Small-angle X-ray scattering (SAXS) for domain arrangements

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

• Specific considerations for pressure studies:

  • Testing pressure sensitivity of individual domains

  • Identification of pressure-sensing domains

  • Analysis of domain interactions under pressure

• Cloning and expression strategies:

  • Design of primers with appropriate restriction sites

  • PCR amplification of domain-encoding sequences

  • Subcloning into expression vectors

  • Expression screening in multiple systems

How does P. profundum DXS interact with other enzymes in the methylerythritol phosphate (MEP) pathway?

Understanding the interactions between P. profundum DXS and other enzymes in the MEP pathway provides insights into pathway regulation and potential metabolic engineering applications:

• MEP pathway organization in P. profundum:

  • Genomic organization of MEP pathway genes

  • Potential operon structures or regulatory clusters

  • Comparative analysis with other bacteria

• Protein-protein interaction studies:

  • Pull-down assays using tagged DXS

  • Bacterial two-hybrid screening

  • Co-immunoprecipitation experiments

  • Cross-linking mass spectrometry

• Metabolic channeling investigation:

  • Kinetic studies for substrate channeling

  • Co-expression of DXS with downstream enzymes

  • Isotope dilution experiments

  • Localization studies of pathway enzymes

• Regulatory interactions:

  • Investigation of feedback inhibition by pathway end products

  • Analysis of allosteric regulation by metabolites

  • Identification of regulatory proteins interacting with DXS

• Pressure-dependent interactions:

  • Studies of protein-protein interactions under varying pressure conditions

  • Investigation of pathway flux control under pressure

  • Analysis of complex formation at different pressures

• Pathway engineering approaches:

  • Co-expression of optimized enzyme combinations

  • Design of synthetic scaffolds to enhance pathway efficiency

  • Metabolic modeling to identify flux control points

• Comparative analysis with other organisms:

  • Comparison with the MEP pathway organization in other bacteria

  • Identification of unique features in P. profundum

  • Analysis of evolutionary adaptations to deep-sea environments

This research area is particularly relevant for understanding how metabolic pathways are adapted to function under extreme conditions and how these adaptations might be harnessed for biotechnological applications.