Recombinant Rhodopirellula baltica 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is Rhodopirellula baltica and how has its taxonomy recently changed?

Rhodopirellula baltica is a marine bacterium belonging to the family Pirellulaceae of the phylum Planctomycetota. Members of this genus are characterized as aerobic, mesophilic chemoheterotrophs. Recent phylogenomic analysis based on 16S rRNA gene sequences and multi-locus sequence analysis has revealed that the genus Rhodopirellula is actually subdivided into four distinct clades . This heterogeneity has been confirmed through multiple genomic indices including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), average amino acid identity (AAI), and percentage of conserved proteins (POCP) . Based on these findings, researchers have proposed reclassifying Rhodopirellula into four genera: Rhodopirellula sensu stricto and three new genera - Aporhodopirellula, Allorhodopirellula, and Neorhodopirellula . This taxonomic revision is important for researchers working with R. baltica to properly contextualize their work in the current classification system.

What is 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and what is its significance?

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway (also known as the methylerythritol phosphate or MEP pathway) for isoprenoid biosynthesis . This enzyme catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) in a NADPH-dependent reaction. The non-mevalonate pathway is present in many bacteria, including R. baltica, as well as in apicomplexan parasites and plant plastids, but is absent in mammals, making DXR an attractive target for antimicrobial drug development . DXR enzymes typically function as homodimers, as demonstrated by structural studies of DXR from various organisms including Toxoplasma gondii .

Why study recombinant R. baltica DXR specifically?

Studying recombinant R. baltica DXR offers several advantages:

• R. baltica is a marine bacterium with unique adaptations to its environment, potentially resulting in enzymes with novel properties or stability characteristics.

• The non-mevalonate pathway in marine bacteria may have evolved differently from terrestrial organisms, offering insights into enzyme evolution and adaptation.

• Understanding R. baltica DXR structure and function can contribute to broader knowledge about isoprenoid biosynthesis across different taxonomic groups.

• As the rate-limiting enzyme in the pathway, DXR represents a control point for isoprenoid production, making it relevant for both basic science and potential biotechnological applications.

What expression systems are most effective for producing recombinant R. baltica DXR?

Based on successful expression of other R. baltica proteins like RB5312, the following expression systems have proven effective:

For R. baltica DXR specifically, cloning the sequence into a vector like pFO4 with BglII/EcoRI restriction sites has shown success with related proteins . The expression construct should include consideration of the native signal peptide, which in R. baltica proteins is typically cleaved (for example, between residues Ala23 and Gln24 in RB5312) . Including an N-terminal His-tag facilitates purification while maintaining enzyme activity.

What purification strategies yield high-purity recombinant R. baltica DXR?

A multi-step purification strategy is recommended:

• Initial capture using Ni-NTA affinity chromatography (for His-tagged constructs)

• Intermediate purification using ion exchange chromatography

• Polishing step with size exclusion chromatography

Typical purification results can achieve:

Buffer optimization is critical, with typical conditions including: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol. Addition of NADPH (0.1-0.5 mM) during purification can enhance stability.

What crystallization conditions have been successful for R. baltica proteins?

While specific crystallization conditions for R. baltica DXR are not directly reported in the provided literature, successful crystallization of another R. baltica protein (RB5312) provides guidance. The hanging-drop vapor-diffusion method yielded crystals belonging to space group P212121, with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 Å, α = β = γ = 90° . These crystals diffracted to 1.8 Å resolution.

For DXR homologs from other organisms, such as T. gondii, crystallization in the presence of the inhibitor fosmidomycin and cofactor NADPH has been successful . A similar approach could be attempted with R. baltica DXR:

How can one analyze the oligomeric state of R. baltica DXR?

Based on studies of DXR from other organisms, R. baltica DXR is expected to function as a homodimer. Multiple techniques can be employed to determine its oligomeric state:

• Size Exclusion Chromatography (SEC): Comparing elution volumes with known standards

• Dynamic Light Scattering (DLS): Measuring particle size distribution

• Small-Angle X-ray Scattering (SAXS): As demonstrated with T. gondii DXR, SAXS analysis can confirm dimeric conformation in solution with parameters like radius of gyration (Rg) and maximum particle dimension (Dmax)

• Native PAGE: Non-denaturing gel electrophoresis to estimate native molecular weight

• Analytical Ultracentrifugation: Sedimentation velocity experiments

A typical SAXS analysis of DXR might yield parameters similar to those found for T. gondii DXR:

What methods are most effective for assessing DXR enzyme activity?

Several complementary approaches can be used to characterize R. baltica DXR activity:

• Spectrophotometric NADPH oxidation assay: Monitoring decrease in absorbance at 340 nm

• Coupled enzyme assays: Linking DXR activity to a secondary reaction with colorimetric output

• LC-MS analysis: Direct measurement of substrate (DXP) consumption and product (MEP) formation

• Radiometric assays: Using 14C-labeled substrates for high sensitivity

Typical reaction conditions for activity assays include:

Expected kinetic parameters for R. baltica DXR based on other bacterial DXR enzymes:

How can one investigate inhibitor binding to R. baltica DXR?

Understanding inhibitor binding is crucial for structure-based drug design. Several approaches can be utilized:

• Enzyme inhibition assays: Determining IC50 and Ki values

• Thermal shift assays: Measuring changes in protein thermal stability upon inhibitor binding

• Isothermal titration calorimetry (ITC): Quantifying binding thermodynamics

• X-ray crystallography: Determining atomic-level details of inhibitor binding mode

• Surface plasmon resonance (SPR): Measuring binding kinetics

Fosmidomycin, a known DXR inhibitor that has been co-crystallized with T. gondii DXR , provides a starting point for inhibitor studies. Other related compounds like fosmidomycin analogues with α-phenyl-β-thia and β-oxa modifications could be tested against R. baltica DXR .

How can molecular dynamics simulations enhance understanding of R. baltica DXR function?

Molecular dynamics (MD) simulations can provide insights into dynamic aspects of DXR function that are not apparent from static crystal structures:

• Conformational flexibility: Identifying mobile regions that may play roles in catalysis

• Substrate binding pathways: Elucidating how substrates enter the active site

• Allosteric mechanisms: Investigating communication between subunits in the dimer

• Water networks: Determining the role of structured water molecules in catalysis

• Loop dynamics: Analyzing flexible loops that may regulate access to the active site

A typical MD simulation protocol would include:

• System preparation with appropriate force fields

• Solvation in explicit water model with physiological ion concentration

• Energy minimization and equilibration

• Production simulations (100 ns - 1 μs)

• Analysis of trajectories for structural and dynamic properties

How can SAXS complement X-ray crystallography for understanding R. baltica DXR?

SAXS offers several advantages when used in conjunction with crystallography:

• Solution structure determination: Confirming that crystal structure reflects solution conformation

• Flexible region modeling: Characterizing regions that may be disordered in crystal structures

• Ligand-induced conformational changes: Detecting large-scale structural changes upon substrate or inhibitor binding

• Oligomeric state verification: Confirming dimeric assembly in solution

The complementary use of SAXS and crystallography for T. gondii DXR has revealed that parts of the protein not visible in crystal structures, such as flexible loops, can be modeled using Ensemble Optimization Method (EOM) . For R. baltica DXR, SAXS could similarly be used to model flexible regions and verify the dimeric conformation observed in related DXR enzymes.

What approaches can be used to engineer R. baltica DXR for enhanced stability or activity?

Protein engineering strategies for R. baltica DXR might include:

• Rational design based on structural information: Targeting specific residues involved in catalysis or substrate binding

• Consensus approach: Introducing residues that are highly conserved across DXR homologs

• Directed evolution: Creating libraries of variants and screening for desired properties

• Computational design: Using algorithms to predict stabilizing mutations

Potential targets for engineering include:

How does R. baltica DXR compare to homologous enzymes from other organisms?

A comparative analysis of DXR enzymes from different organisms reveals both conserved features and adaptations specific to each organism's environment:

Sequence conservation typically centers around the active site and NADPH binding regions, while major differences are often found in surface loops and N/C-terminal regions.

What insights can be gained from studying the evolution of DXR across the newly reclassified Rhodopirellula genera?

With the recent reclassification of Rhodopirellula into four distinct genera (Rhodopirellula sensu stricto, Aporhodopirellula, Allorhodopirellula, and Neorhodopirellula) , comparative analysis of DXR across these related genera could reveal:

• Conservation of catalytic mechanism despite taxonomic divergence

• Adaptive changes reflecting different ecological niches

• Variations in enzyme kinetics or substrate specificity

• Different responses to potential inhibitors

What strategies can overcome low expression or poor solubility of recombinant R. baltica DXR?

Researchers encountering expression or solubility issues might consider:

How can researchers address challenges in crystallizing R. baltica DXR?

Crystallization challenges can be approached through:

• Protein engineering: Creating truncated constructs or surface entropy reduction mutants

• Complex formation: Co-crystallization with ligands (substrates, inhibitors, cofactors)

• Screening optimization: Using sparse matrix and grid screens with varying protein concentrations

• Seeding techniques: Microseed matrix screening to promote crystal nucleation

• Alternative crystallization methods: Lipidic cubic phase for membrane-associated forms, counter-diffusion methods

The successful crystallization of the R. baltica protein RB5312 using the hanging-drop vapor-diffusion method provides a starting point, but extensive screening may be necessary to identify optimal conditions for DXR.