Recombinant Acinetobacter sp. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is the biochemical role of DXR in bacterial metabolism?

DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) catalyzes the second step in the non-mevalonate pathway for isoprenoid biosynthesis, which is essential for bacterial survival. The enzyme converts 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP) in a NADPH-dependent reaction. This pathway is crucial for producing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are precursors for all isoprenoid compounds necessary for bacterial cell membrane and various metabolic functions .

Why is Acinetobacter sp. DXR considered a valuable antibiotic target?

Acinetobacter sp. DXR is considered a valuable antibiotic target because humans exclusively use the mevalonate pathway for isoprenoid biosynthesis rather than the non-mevalonate pathway used by many pathogenic bacteria. This fundamental biochemical difference creates an opportunity for selective targeting of bacterial metabolism without affecting human enzymes, potentially reducing side effects. The essentiality of this enzyme for bacterial survival combined with its absence in humans makes it an attractive target for developing novel antibiotics to combat drug resistance in pathogenic bacteria .

What expression systems are most effective for recombinant Acinetobacter sp. DXR?

For recombinant Acinetobacter sp. DXR expression, E. coli-based systems are typically most effective, utilizing vectors containing T7 promoters. The methodology employed for successful expression includes optimization of induction conditions (IPTG concentration, temperature, and duration) and consideration of codon optimization. Similar to approaches used with ACCase from Acinetobacter baumannii, researchers should express the enzyme with an affinity tag (typically His-tag) to facilitate purification, incubate cultures at 30°C rather than 37°C after induction to enhance soluble protein yield, and supplement with appropriate cofactors if needed .

What purification strategy yields the highest purity and activity for recombinant DXR?

A multi-step purification strategy typically yields the highest purity and activity for recombinant DXR. This approach typically begins with immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture His-tagged DXR, followed by ion-exchange chromatography to remove residual contaminants, and size-exclusion chromatography as a final polishing step. To preserve enzymatic activity, all purification steps should be performed at 4°C with buffers containing stabilizing agents such as DTT or β-mercaptoethanol, glycerol (10-15%), and potentially EDTA to prevent metal-catalyzed oxidation. Quality assessment should include SDS-PAGE and enzymatic activity measurements using malonyl-CoA formation detection methods similar to those employed for ACCase purification .

How can researchers optimize protein folding and solubility when expressing recombinant DXR?

To optimize protein folding and solubility of recombinant DXR, researchers should systematically investigate multiple parameters: (1) Lower induction temperatures (16-25°C) often promote proper folding over expression rate; (2) Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ) can significantly improve folding efficiency; (3) Fusion partners such as MBP (maltose-binding protein) or SUMO can enhance solubility; (4) Buffer optimization during lysis and purification should include testing various pH values (typically pH 7.0-8.5), salt concentrations (100-500 mM NaCl), and solubility enhancers (0.1-1% Triton X-100, 0.5-2 M urea, or 5-10% glycerol). If inclusion bodies form despite these measures, controlled denaturation followed by step-wise refolding using dialysis against decreasing concentrations of denaturants may be necessary.

What are the established methods for measuring DXR enzyme activity?

Several established methods exist for measuring DXR enzyme activity. The most direct approach involves monitoring the conversion of DXP to MEP using liquid chromatography-tandem mass spectrometry (LC-MS/MS), similar to the detection of malonyl-CoA formation in ACCase activity assays . Additionally, researchers can employ spectrophotometric assays that monitor NADPH oxidation at 340 nm, as DXR uses NADPH as a cofactor for reduction. For higher throughput screening, particularly in inhibitor discovery, luminescence-based assays that monitor ATP depletion (if applicable to the specific reaction conditions) or coupling the reaction with other enzymes that produce detectable signals may be implemented. Each method has specific advantages depending on the research question, with LC-MS/MS offering the highest specificity but requiring specialized equipment.

How should kinetic parameters for DXR be determined and validated?

Kinetic parameters for DXR should be determined through steady-state kinetics experiments following Michaelis-Menten principles. The methodology involves measuring initial reaction velocities at varying substrate concentrations while keeping enzyme concentration constant. For complete characterization, researchers should determine k(cat) and K(M) values for both substrates (DXP and NADPH) using either fixed-concentration or matrix approaches. Validation requires technical replicates (minimum of 3), statistical analysis of curve fitting (R² values > 0.95), and confirmation of linear enzyme concentration dependence. Additionally, comparing kinetic parameters with published values for DXR from related species provides context for interpretation. When analyzing inhibitors, IC₅₀ determination should follow similar methodological rigor with appropriate controls to ensure the validity of measurements .

What factors most significantly impact DXR activity measurements in vitro?

Several factors significantly impact DXR activity measurements in vitro. Buffer composition is crucial, with pH typically maintained between 7.0-8.0 and including divalent metal ions (Mg²⁺ or Mn²⁺) as cofactors. Reducing agents (DTT, β-mercaptoethanol) are essential to prevent oxidation of critical cysteine residues. Temperature control is vital, with most assays conducted at 25-37°C, and temperature fluctuations of even 2-3°C can significantly alter activity measurements. Enzyme stability during the assay timeframe should be verified through progress curve linearity tests. Substrate purity is particularly important, as commercial DXP preparations may contain impurities that inhibit the enzyme. Finally, researchers must account for potential substrate or product inhibition effects by validating linearity of reaction progress over the entire measurement period.

What structural features distinguish Acinetobacter sp. DXR from other bacterial DXRs?

While specific structural information for Acinetobacter sp. DXR is limited in the provided sources, comparative analysis with other bacterial DXRs suggests several distinguishing features may exist. Based on structural studies of related enzymes, Acinetobacter sp. DXR likely contains conserved catalytic domains with species-specific variations in flexible loop regions that contribute to substrate binding pocket differences. These structural variations may influence inhibitor binding profiles and catalytic efficiency. Understanding these distinctions requires techniques such as X-ray crystallography or cryo-electron microscopy to directly compare Acinetobacter sp. DXR with well-characterized DXRs from organisms like E. coli, for which structural information is more readily available .

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) inform our understanding of DXR dynamics?

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides crucial insights into DXR dynamics by measuring the rate of hydrogen-deuterium exchange in different protein regions. As demonstrated with the related enzyme DXPS, HDX-MS can reveal regions of high flexibility and conformational changes upon substrate binding . For DXR research, this approach can identify: (1) dynamic regions involved in substrate recognition and catalysis; (2) conformational changes induced by inhibitor binding; (3) allosteric communication networks within the protein; and (4) potential target sites for novel inhibitor design. The methodology requires careful experimental design, including optimized peptide digestion conditions (as shown in the DXPS study's extensive peptide mapping table), controlled deuterium exchange times, and rigorous data analysis to distinguish EX1 (cooperative unfolding) from EX2 (localized fluctuations) exchange behaviors .

What advanced computational approaches best predict DXR conformational changes during catalysis?

Advanced computational approaches for predicting DXR conformational changes during catalysis include molecular dynamics (MD) simulations with enhanced sampling techniques, normal mode analysis, and machine learning-based methods. Long-timescale (microsecond to millisecond) MD simulations can capture conformational transitions between open and closed states as observed in related enzymes like DXPS . To achieve comprehensive sampling, researchers should implement techniques such as replica exchange, metadynamics, or accelerated MD. These approaches should incorporate explicit solvent models and employ appropriate force fields validated for metalloenzymes. Integration of experimental data (e.g., HDX-MS results) with computational predictions through methods like ensemble refinement provides the most robust understanding of DXR's conformational landscape during different stages of catalysis.

What is the mechanism of action for fosmidomycin against DXR?

Fosmidomycin acts as a competitive inhibitor of DXR by mimicking the substrate DXP. The phosphonate group of fosmidomycin mimics the phosphate group of DXP, while the hydroxamate group chelates the divalent metal ion in the active site, disrupting the enzyme's catalytic function. This mechanism prevents the conversion of DXP to MEP, thereby inhibiting isoprenoid biosynthesis essential for bacterial survival. Despite being a potent inhibitor against many Gram-negative bacteria and showing antimalarial activity in clinical trials, fosmidomycin has limitations including poor pharmacokinetics with a short half-life (0.5-1.5 hours) and ineffectiveness against certain pathogens including Gram-positive bacteria and some parasites .

How can high-throughput screening approaches be optimized for identifying novel DXR inhibitors?

Optimization of high-throughput screening (HTS) approaches for identifying novel DXR inhibitors requires a multi-faceted strategy. Researchers should develop luminescence-based assays that monitor ATP depletion or other coupling reactions that produce easily detectable signals, similar to methods developed for ACCase inhibitor screening . Compound libraries should be rationally designed based on structure-guided approaches, focusing on scaffolds distinct from fosmidomycin to overcome existing resistance mechanisms. The screening cascade should include: (1) Primary screening using recombinant E. coli or Acinetobacter sp. DXR enzymes; (2) Secondary confirmation assays with orthogonal detection methods; (3) Counter-screening against human enzymes to ensure selectivity; (4) Evaluation of physicochemical properties; and (5) Early assessment of antibacterial activity against fosmidomycin-resistant organisms. This comprehensive approach has successfully identified novel DXR inhibitors with Ki values as low as 310 nM .

What structure-activity relationships have been established for DXR inhibitors beyond fosmidomycin?

Structure-activity relationship (SAR) studies for DXR inhibitors beyond fosmidomycin have revealed several important pharmacophore features. Based on rational structure-based design approaches, novel drug-like inhibitors with Ki values as low as 310 nM against E. coli DXR have been developed . These compounds bind to DXR in modes distinct from fosmidomycin, offering potential advantages for overcoming resistance. Key SAR findings include: (1) The importance of metal-chelating groups that interact with the active site divalent cation; (2) Hydrophobic moieties that enhance binding through interactions with nonpolar pockets; (3) Hydrogen bond donors/acceptors that form specific interactions with conserved residues; and (4) Appropriate spacing between these key functional groups. Optimization of these features through medicinal chemistry and computational modeling has led to the development of compound libraries with improved activity profiles .

How does Acinetobacter sp. DXR compare functionally to DXR enzymes from other pathogens?

While specific comparative data for Acinetobacter sp. DXR is limited in the provided sources, functional comparisons with DXR enzymes from other pathogens would likely reveal both conservation of catalytic mechanism and species-specific variations. Based on studies of related enzymes, Acinetobacter sp. DXR probably shares the core NADPH-dependent reduction mechanism but may exhibit differences in substrate affinity, catalytic efficiency, and inhibitor sensitivity. These differences could result from variations in active site architecture or allosteric regulation mechanisms. Similar comparative studies of ACCase from Acinetobacter baumannii and Klebsiella pneumoniae revealed comparable kinetic parameters (k(cat) and K(M) values) and inhibitor sensitivity despite being from different bacterial species .

What are the most common challenges in expressing active recombinant DXR?

The most common challenges in expressing active recombinant DXR include protein insolubility, improper folding, and loss of activity during purification. These issues often stem from the complex nature of DXR, which requires proper cofactor binding and specific structural arrangements for activity. To address insolubility, researchers should optimize expression conditions by testing different E. coli strains (BL21(DE3), Rosetta, ArcticExpress), varying induction parameters (lower IPTG concentrations of 0.1-0.5 mM, reduced temperatures of 16-25°C), and exploring fusion tags (MBP, SUMO) that enhance solubility. Improper folding may be mitigated through co-expression with chaperones or addition of stabilizing agents (glycerol, reducing agents) to lysis buffers. Loss of activity during purification can be minimized by including cofactors in purification buffers and limiting exposure to potentially denaturing conditions.

How can researchers troubleshoot inconsistent DXR activity assay results?

Inconsistent DXR activity assay results can be troubleshooted through a systematic approach addressing multiple variables. First, enzyme stability should be verified through thermal shift assays to ensure the protein remains folded during the experiment. Second, buffer components should be freshly prepared and precisely pH-adjusted, as small variations can significantly impact activity. Third, substrate quality should be confirmed through analytical methods (HPLC, NMR) to detect potential degradation or contamination. Fourth, the linearity of the assay should be established by varying enzyme concentrations and measurement times. Fifth, the potential for product inhibition should be investigated by monitoring reaction progress. Similar troubleshooting approaches have been successfully applied to ACCase enzyme activity assays, where direct detection of malonyl-CoA formation by LC-MS/MS provided reliable and reproducible results .

What strategies can overcome challenges in crystallizing DXR for structural studies?

Crystallizing DXR for structural studies presents several challenges that can be overcome through systematic optimization strategies. Researchers should employ a multi-faceted approach including: (1) Protein engineering to remove flexible regions that may hinder crystallization, guided by HDX-MS data similar to that obtained for DXPS ; (2) Screening diverse crystallization conditions using sparse matrix approaches followed by fine grid optimization; (3) Co-crystallization with substrates, products, or inhibitors to stabilize specific conformations; (4) Testing various truncation constructs to identify minimal functional domains; (5) Surface entropy reduction through mutation of high-entropy surface residues to alanine; and (6) Employing microseed matrix screening to promote crystal nucleation. Additionally, alternative structural determination methods such as cryo-EM may be considered if crystallization proves persistently challenging, particularly for capturing dynamic conformational states.

What emerging technologies show promise for enhancing DXR research?

Several emerging technologies show significant promise for enhancing DXR research. CRISPR-Cas9 gene editing technologies enable precise genomic modifications to study DXR function in vivo, creating conditional knockdowns to assess essentiality under various conditions. Cryo-electron microscopy advancements now allow visualization of conformational ensembles without crystallization, potentially revealing dynamic states similar to those observed in DXPS . Microfluidic enzyme assay platforms can dramatically increase throughput while reducing reagent consumption for inhibitor screening. AlphaFold2 and related AI-based structural prediction tools may help model species-specific DXR variants where experimental structures are unavailable. Time-resolved HDX-MS techniques can capture transient conformational changes during catalysis with millisecond resolution. Finally, chemoproteomics approaches using activity-based probes can assess target engagement of inhibitors in complex biological settings.

How might research on Acinetobacter sp. DXR contribute to addressing antimicrobial resistance?

Research on Acinetobacter sp. DXR has significant potential to address antimicrobial resistance through multiple avenues. As an essential enzyme absent in humans but present in many pathogens, DXR represents an attractive target for developing novel antibiotics with potentially reduced side effects . Understanding the specific structural and functional properties of Acinetobacter sp. DXR can guide the development of species-selective inhibitors that could be particularly valuable against multidrug-resistant Acinetobacter species. The development of DXR inhibitors with mechanisms distinct from fosmidomycin offers opportunities to overcome existing resistance mechanisms. Furthermore, combination approaches targeting multiple enzymes in the non-mevalonate pathway could create synergistic effects that reduce the likelihood of resistance development. This multi-target approach parallels strategies successfully employed in other therapeutic areas to combat resistance evolution.

What are the most significant unresolved questions regarding DXR catalytic mechanism?

Several significant unresolved questions regarding DXR catalytic mechanism warrant further investigation. The precise order of substrate binding (DXP and NADPH) and product release remains incompletely characterized, requiring detailed pre-steady-state kinetic analyses. The role of conformational dynamics in catalysis, particularly whether DXR undergoes substantial domain movements similar to DXPS , requires clarification through techniques like HDX-MS and single-molecule FRET. The specific residues involved in acid-base catalysis during the isomerization step and their protonation states need definitive assignment through pH-dependent kinetics and targeted mutagenesis. The potential for allosteric regulation of DXR activity by pathway intermediates or other cellular metabolites remains largely unexplored. Finally, species-specific variations in catalytic mechanism that might explain differences in inhibitor sensitivity between Acinetobacter sp. DXR and DXR from other pathogens require comparative enzymological studies.