Recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase (cdh)

What is CDP-diacylglycerol pyrophosphatase (cdh) and what is its function in Mycobacterium bovis?

CDP-diacylglycerol pyrophosphatase (cdh) is an enzyme (EC 3.6.1.26) also known as CDP-diacylglycerol phosphatidylhydrolase or CDP-diglyceride hydrolase . This enzyme plays a crucial role in phospholipid metabolism in mycobacteria by catalyzing the hydrolysis of CDP-diacylglycerol, which is a key intermediate in phospholipid biosynthesis.

In Mycobacterium bovis, this enzyme is particularly important because it regulates the availability of CDP-diacylglycerol (CDP-DAG), which serves as the central liponucleotide intermediate for phospholipid biosynthesis . CDP-DAG is essential for the synthesis of phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL), which are critical components of the mycobacterial cell membrane and cell wall structure .

How is CDP-diacylglycerol synthesized and what is its significance in the phospholipid biosynthetic pathway?

CDP-diacylglycerol synthesis follows a specific pathway in mycobacteria:

• Phosphatidic acid (PA) is first generated through the acylation of glycerol-3-phosphate, which is the initiating event in phospholipid biosynthesis .

• PA is then converted to CDP-DAG by the enzyme CTP:phosphatidate cytidylyltransferase (also known as CDS or CDP-diacylglycerol synthase) .

• This reaction utilizes cytidine triphosphate (CTP) as a substrate, producing CDP-DAG as the key intermediate .

The significance of CDP-DAG in phospholipid biosynthesis is profound:

• In bacteria including mycobacteria, CDP-DAG serves as the precursor for the biosynthesis of all major phospholipids including phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), and phosphatidylethanolamine (PE) .

• In mycobacteria specifically, CDP-DAG is crucial for the synthesis of phosphatidylinositol (PI), which acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside (PIM), lipomannan (LM), and lipoarabinomannan (LAM) .

What are the optimal storage conditions for recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase?

For optimal activity and stability of recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase, the following storage conditions are recommended:

• Long-term storage: The protein should be stored at -20°C or -80°C. The shelf life is approximately 6 months for liquid form and 12 months for lyophilized form under these conditions .

• Working aliquots: These can be stored at 4°C for up to one week .

• Reconstitution: The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• For long-term storage after reconstitution: Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and aliquot before storing at -20°C/-80°C .

• Avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity and activity .

How does CDP-diacylglycerol pyrophosphatase activity impact mycobacterial cell wall synthesis and pathogenicity?

CDP-diacylglycerol pyrophosphatase activity directly influences the availability of phosphatidylinositol (PI), which serves as a critical lipid anchor for essential components of the mycobacterial cell wall. This impact operates through several mechanisms:

• Regulation of PI production: By hydrolyzing CDP-DAG, this enzyme modulates the availability of this key substrate for PI synthesis, affecting the subsequent production of phosphatidylinositol-phosphate (PIP), which is then dephosphorylated to PI .

• Cell wall integrity: The glycolipids anchored by PI—particularly phosphatidylinositol mannoside (PIM), lipomannan (LM), and lipoarabinomannan (LAM)—are crucial structural elements that maintain cell wall integrity and permeability .

• Virulence modulation: In Mycobacterium tuberculosis (closely related to M. bovis), these PI-anchored glycolipids function as important virulence factors that modulate the host immune response . Alterations in cdh activity could therefore potentially impact pathogenicity.

• Essential nature: The pathway for PI biosynthesis has been demonstrated to be of vital importance for growth and viability of mycobacteria, with disruption of this pathway significantly compromising bacterial survival .

Researchers investigating this enzyme should consider these multiple roles when designing experiments to study its function or when considering it as a potential drug target.

What methodological approaches are most effective for assaying CDP-diacylglycerol pyrophosphatase activity in vitro?

For effective in vitro assessment of CDP-diacylglycerol pyrophosphatase activity, researchers should consider the following methodological approaches:

• Radiometric assays:

  • Substrate preparation: Use radiolabeled CDP-DAG (typically 14C or 3H labeled)

  • Reaction conditions: Buffer containing 50-100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 0.1% Triton X-100

  • Detection: Separate reaction products by thin-layer chromatography and quantify using liquid scintillation counting

• Coupled enzyme assays:

  • Principle: Link the release of CMP or pyrophosphate to a colorimetric or fluorometric indicator reaction

  • Components needed: Auxiliary enzymes (such as pyrophosphatase and phosphodiesterase), alongside appropriate chromogenic substrates

  • Advantages: Real-time monitoring of reaction kinetics

• HPLC-based analysis:

  • Sample preparation: Extract lipids using chloroform:methanol (2:1, v/v)

  • Separation: Reverse-phase HPLC with appropriate solvent gradient

  • Detection: UV absorbance at 254 nm for detection of the cytidine moiety

• Considerations for experimental design:

  • Enzyme purity should be >85% as assessed by SDS-PAGE

  • Inclusion of divalent cations (Mg2+ or Mn2+) is essential for optimal activity

  • pH optimization is crucial, with most assays performed in the pH range of 7.0-7.5

  • Temperature control at 37°C for physiologically relevant activity measurements

These methodological approaches can be adapted based on specific research questions and available laboratory equipment.

How does the structure of mycobacterial CDP-diacylglycerol pyrophosphatase compare to homologous enzymes in other bacterial species?

The structural comparison between mycobacterial CDP-diacylglycerol pyrophosphatase and its homologs in other bacterial species reveals important evolutionary and functional insights:

• Conserved catalytic domains:

  • The CDP-alcohol phosphotransferase domain is highly conserved across bacterial species, containing the eight amino acid signature motif (D1xxD2G1xxAR...G2xxxD3xxxD4) involved in cytidine-diphosphate and metal binding, as well as in catalysis .

  • This conservation suggests fundamental mechanistic similarities in CDP-DAG metabolism across bacterial kingdoms.

• Mycobacteria-specific structural features:

  • Mycobacterial CDP-diacylglycerol pyrophosphatases possess unique transmembrane topology with specific membrane-binding domains that facilitate interaction with the mycobacterial cell envelope.

  • These structural adaptations likely reflect the specialized cell wall architecture of mycobacteria.

• Substrate binding pocket variations:

  • Comparative analysis of crystallographic data reveals species-specific differences in the substrate binding pocket, particularly in regions accommodating the diacylglycerol portion of CDP-DAG.

  • These variations may contribute to differences in substrate specificity and catalytic efficiency.

• Metal coordination sites:

  • Divalent cation binding sites are structurally conserved but show subtle differences in coordination geometry between mycobacterial enzymes and other bacterial homologs.

  • These differences may influence metal preference and catalytic rates.

Researchers should note that while structural conservation exists in the catalytic core, the regulatory domains of these enzymes often show greater divergence, potentially reflecting different metabolic control mechanisms across bacterial species.

What are the challenges and strategies for crystallization of Mycobacterium bovis CDP-diacylglycerol pyrophosphatase for structural studies?

Crystallization of Mycobacterium bovis CDP-diacylglycerol pyrophosphatase presents several challenges that researchers must overcome to obtain high-quality crystals for structural determination:

• Membrane protein challenges:

  • CDP-diacylglycerol pyrophosphatase is an integral membrane protein, making it inherently difficult to crystallize using conventional methods.

  • Strategy: Use of detergents like n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) for solubilization, followed by lipidic cubic phase (LCP) crystallization techniques.

• Protein stability issues:

  • The enzyme may have limited stability once extracted from its native membrane environment.

  • Strategy: Addition of stabilizing agents such as glycerol (5-50%) and specific lipids to maintain native-like folding .

• Construct optimization:

  • Full-length constructs may have flexible regions that inhibit crystal formation.

  • Strategy: Engineering truncated constructs or fusion proteins with crystallization chaperones, similar to the approach used for MkPIPS where an Archaeoglobus fulgidus CTD fusion protein facilitated crystallization .

• Crystallization conditions:

  • Systematic screening of crystallization conditions with various precipitants, buffers, and additives is essential.

  • Strategy: High-throughput crystallization screening followed by optimization of promising conditions.

• Co-crystallization with substrates:

  • Co-crystallization with CDP or substrate analogs can stabilize the protein and provide valuable functional insights.

  • Strategy: Use of non-hydrolyzable substrate analogs to capture enzyme-substrate complexes without catalytic turnover, as demonstrated with MkPIPS .

Recent successes with related enzymes like PIPS from Mycobacterium kansasii suggest that a multi-pronged approach combining structural genomics, crystallization chaperones, and crystal engineering offers the best chance for successful structural determination .

How do structural variations in CDP-diacylglycerol pyrophosphatase affect substrate specificity and catalytic efficiency?

The structure-function relationship in CDP-diacylglycerol pyrophosphatase reveals how specific structural elements influence substrate recognition and catalytic properties:

• Active site architecture:

  • The enzyme contains a conserved CDP-alcohol phosphotransferase domain with the characteristic signature motif (D1xxD2G1xxAR...G2xxxD3xxxD4) .

  • The spatial arrangement of these residues creates a pocket that accommodates the CDP-diacylglycerol substrate with high specificity.

  • Mutations in this region significantly alter catalytic efficiency and substrate preference.

• Transmembrane domains and substrate access:

  • As an integral membrane protein, the enzyme's transmembrane helices create a hydrophobic environment that facilitates interaction with the lipid portions of the substrate.

  • The arrangement of these helices forms a lateral access pathway for substrates from the membrane bilayer.

• Metal coordination site:

  • The enzyme requires divalent cations (typically Mg2+ or Mn2+) for catalysis.

  • The coordination geometry of the metal-binding site influences both substrate positioning and the efficiency of the catalytic reaction.

• Substrate-induced conformational changes:

  • Binding of CDP-diacylglycerol induces conformational changes, particularly around the transmembrane helix 2 (TM2) region, as observed in the related enzyme MtPgsA1 .

  • These conformational changes are critical for proper substrate alignment and catalysis.

This structure-function relationship is particularly relevant for researchers developing inhibitors targeting this enzyme or engineering variants with altered catalytic properties.

What role does CDP-diacylglycerol pyrophosphatase play in phospholipid remodeling during mycobacterial adaptation to environmental stresses?

CDP-diacylglycerol pyrophosphatase (cdh) plays a critical role in phospholipid remodeling during mycobacterial adaptation to environmental stresses:

• Stress-responsive regulation:

  • Under various environmental stresses (pH changes, oxidative stress, nutrient limitation), mycobacteria modify their membrane phospholipid composition for survival.

  • CDP-diacylglycerol pyrophosphatase activity has been shown to respond to these stress conditions, with altered expression levels observed under specific stress scenarios.

• Phospholipid homeostasis:

  • By regulating CDP-DAG levels, this enzyme influences the balance between different phospholipid biosynthetic pathways.

  • During stress adaptation, shifts in this balance can redirect phospholipid synthesis toward specific lipid classes that enhance stress resistance.

• Cell envelope remodeling:

  • Environmental stresses often require remodeling of the mycobacterial cell envelope, including changes in the phosphatidylinositol-derived glycolipids.

  • CDP-diacylglycerol pyrophosphatase activity affects the availability of precursors for these modifications.

• Metabolic integration:

  • The enzyme functions at a critical junction point in phospholipid metabolism, allowing for integration of signals from various metabolic pathways.

  • This position enables it to coordinate lipid synthesis with other cellular responses to stress.

Research examining the enzyme's role in stress adaptation typically employs gene expression analysis, lipidomics profiling, and phenotypic characterization of mutant strains with altered cdh activity under defined stress conditions.

How can structural knowledge of CDP-diacylglycerol pyrophosphatase inform drug design strategies against Mycobacterium tuberculosis and related pathogenic mycobacteria?

Structural insights into CDP-diacylglycerol pyrophosphatase provide valuable guidance for rational drug design targeting mycobacterial pathogens:

• Unique structural features for selectivity:

  • The prokaryotic PI synthesis pathway is unique to mycobacteria and a few other bacterial species, distinct from the eukaryotic pathway .

  • This pathway difference, particularly the use of inositol-phosphate rather than myo-inositol as a substrate, provides an opportunity for selective targeting without affecting host enzymes.

• Essential enzyme targeting:

  • Phosphatidylinositol biosynthesis is essential for mycobacterial viability, as demonstrated through conditional knockout studies .

  • This essentiality makes cdh and related enzymes in this pathway attractive drug targets.

• Structure-based inhibitor design approaches:

  • Crystal structures reveal specific binding pockets that can be exploited for inhibitor design.

  • The CDP-binding site offers opportunities for nucleotide analog inhibitors.

  • The inositol-phosphate binding pocket provides another target site for competitive inhibitors .

• Rational design strategies:

  • Fragment-based drug discovery can identify initial binding compounds for further optimization.

  • Structure-guided modifications can improve inhibitor potency and selectivity.

  • In silico screening against the crystal structure can identify potential inhibitors from compound libraries.

• Drug resistance considerations:

  • Structural analysis of potential resistance mutations can inform inhibitor design to minimize resistance development.

  • Targeting highly conserved regions of the enzyme reduces the likelihood of viable resistance mutations.

This structural knowledge, combined with biochemical characterization, provides a solid foundation for developing novel antimycobacterial agents with potentially lower rates of resistance development compared to current therapies.

What expression systems are most effective for producing active recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase for functional studies?

Several expression systems have been evaluated for the production of active recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase, each with distinct advantages and limitations:

• E. coli expression system:

  • Most commonly used system for initial production attempts .

  • Advantages: Rapid growth, high yields, well-established protocols, and cost-effectiveness.

  • Challenges: Potential misfolding of membrane proteins, formation of inclusion bodies, and lack of mycobacterial-specific post-translational modifications.

  • Optimization strategies: Use of specialized E. coli strains (C41(DE3), C43(DE3), Rosetta), fusion tags (MBP, SUMO), lower induction temperatures (16-20°C), and mild detergents for extraction.

• Mycobacterial expression systems:

  • Expression in non-pathogenic mycobacteria like M. smegmatis.

  • Advantages: Native-like membrane environment and post-translational modifications, correct folding machinery.

  • Challenges: Slower growth, lower yields, and more complex extraction procedures.

  • Best used when authentic enzyme function is critical for the research question.

• Cell-free expression systems:

  • Emerging approach for membrane protein production.

  • Advantages: Rapid production, ability to directly incorporate detergents or lipids.

  • Challenges: Lower yields, higher cost, and potential for incomplete folding.

For most functional studies, E. coli expression with appropriate optimization represents the best balance of yield, activity, and technical feasibility, as evidenced by the successful production of related mycobacterial proteins with >85% purity .

What are the critical parameters for assessing the quality and activity of purified recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase?

To ensure high-quality and functionally active recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase for research applications, several critical quality control parameters should be assessed:

• Purity assessment:

  • SDS-PAGE analysis: A minimum purity of >85% is recommended for functional studies .

  • Size exclusion chromatography: To verify monodispersity and absence of aggregates.

  • Mass spectrometry: For accurate molecular weight confirmation and detection of potential modifications.

• Structural integrity:

  • Circular dichroism (CD) spectroscopy: To confirm proper secondary structure.

  • Thermal shift assays: To assess protein stability and proper folding.

  • Limited proteolysis: To verify correct folding by examining protease accessibility patterns.

• Functional activity:

  • Enzyme kinetics determination: Measuring Km, Vmax, and kcat values using appropriate substrate concentrations.

  • Specific activity calculation: Typically expressed as μmol product formed per minute per mg protein.

  • Metal dependency: Verification of activity enhancement with divalent cations (Mg2+ or Mn2+).

• Storage stability assessment:

  • Activity retention after storage at different temperatures (-80°C, -20°C, 4°C).

  • Effect of freeze-thaw cycles on enzyme activity.

  • Stability in different buffer compositions and with various additives (glycerol, reducing agents) .

• Detergent compatibility:

  • Activity assessment in the presence of different detergents used for solubilization.

  • Verification that the detergent micelle size does not interfere with enzyme function.

Researchers should document these parameters thoroughly to ensure reproducibility and reliability of subsequent experiments using the purified enzyme.

What approaches can be used to study the interaction between CDP-diacylglycerol pyrophosphatase and other enzymes in the phospholipid biosynthetic pathway?

Understanding the interactions between CDP-diacylglycerol pyrophosphatase and other enzymes in the phospholipid biosynthetic pathway requires specialized methodological approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP): To identify native protein complexes involving cdh in mycobacterial lysates.

  • Bacterial two-hybrid assays: For screening potential interaction partners.

  • Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics and affinity.

  • Förster resonance energy transfer (FRET): For detecting proximity between cdh and other enzymes when tagged with appropriate fluorophores.

• Metabolic flux analysis:

  • Isotope labeling: Using stable isotope-labeled precursors to track metabolic flux through the pathway.

  • Tandem mass spectrometry: For detection and quantification of labeled intermediates.

  • Mathematical modeling: To integrate experimental data and predict pathway dynamics.

• Genetic approaches:

  • Conditional knockdowns: To assess the impact of cdh depletion on other pathway enzymes.

  • Synthetic genetic arrays: To identify genetic interactions suggestive of functional relationships.

  • Promoter reporter fusions: To study coordinated expression of pathway enzymes.

• Structural biology techniques:

  • Cryo-electron microscopy: For visualization of multi-enzyme complexes.

  • Cross-linking mass spectrometry: To identify interaction interfaces between cdh and partner proteins.

  • Hydrogen-deuterium exchange mass spectrometry: To map interaction surfaces.

• Systems biology integration:

  • Multi-omics data integration: Combining transcriptomics, proteomics, and lipidomics data.

  • Network analysis: To position cdh within the broader metabolic network.

  • Perturbation studies: Examining system-wide effects of cdh modulation.

These approaches can be combined to build a comprehensive understanding of how cdh functions within the context of the entire phospholipid biosynthetic pathway in mycobacteria.

How can recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase be used to screen for potential inhibitors as tuberculosis drug candidates?

Recombinant Mycobacterium bovis CDP-diacylglycerol pyrophosphatase provides an excellent platform for inhibitor screening in tuberculosis drug discovery pipelines:

• High-throughput screening (HTS) assay development:

  • Enzymatic activity-based screening: Using purified recombinant enzyme (>85% purity) to measure inhibition of CDP-DAG hydrolysis.

  • Fluorescence-based assays: Development of FRET or fluorogenic substrate-based assays for rapid screening.

  • Coupled enzyme assays: Linking cdh activity to easily detectable enzymatic reactions for HTS compatibility.

• Structure-based virtual screening:

  • In silico docking: Using available crystal structures of related CDP-alcohol phosphotransferases like PIPS to predict binding of virtual compound libraries.

  • Pharmacophore modeling: Developing models based on known substrates and inhibitors.

  • Fragment-based approaches: Identifying small molecular fragments that bind to different regions of the enzyme.

• Validation of hit compounds:

  • IC50 determination: Dose-response studies to determine inhibitory potency.

  • Mechanism of inhibition studies: Kinetic analysis to determine competitive, non-competitive, or uncompetitive inhibition.

  • Binding affinity measurements: Using techniques like isothermal titration calorimetry (ITC) or microscale thermophoresis (MST).

• Secondary screening cascade:

  • Selectivity profiling: Testing activity against human homologs to ensure selectivity.

  • Mycobacterial growth inhibition: Assessing whole-cell activity against M. bovis BCG and M. tuberculosis.

  • Cytotoxicity assessment: Determining safety profile using mammalian cell lines.

• Medicinal chemistry optimization:

  • Structure-activity relationship (SAR) studies: Using the recombinant enzyme to guide chemical modifications of hit compounds.

  • Physiochemical property optimization: Improving parameters like solubility and permeability while maintaining target engagement.

This systematic approach leveraging recombinant cdh can significantly accelerate the discovery of novel anti-tuberculosis compounds targeting this essential pathway.

What insights can comparative studies of CDP-diacylglycerol pyrophosphatase across different mycobacterial species provide for understanding phylogenetic relationships and evolutionary adaptations?

Comparative analysis of CDP-diacylglycerol pyrophosphatase across mycobacterial species offers valuable insights into evolutionary relationships and adaptations:

• Sequence conservation patterns:

  • Core catalytic domains: The CDP-alcohol phosphotransferase signature motif (D1xxD2G1xxAR...G2xxxD3xxxD4) shows high conservation across species, reflecting functional constraints .

  • Variable regions: Differences in non-catalytic regions may reflect species-specific adaptations to environmental niches.

  • Phylogenetic analysis: Sequence divergence patterns align with established mycobacterial evolutionary trees, with closer relatedness between M. tuberculosis and M. bovis compared to non-pathogenic species.

• Substrate specificity evolution:

  • Species-specific variations in substrate binding regions correlate with differences in membrane phospholipid composition.

  • These variations may reflect adaptation to different host environments or free-living conditions.

  • For example, M. kansasii PIPS shows 86% identity to M. tuberculosis ortholog but exhibits distinct catalytic properties .

• Regulatory element diversity:

  • Promoter region analysis reveals differences in transcriptional regulation mechanisms.

  • These differences may indicate evolutionary adaptations to various growth conditions and stress responses.

  • For instance, pathogenic species show more complex regulation patterns compared to environmental mycobacteria.

• Structural adaptations:

  • Comparative structural biology reveals species-specific differences in protein folding and stability.

  • These adaptations may reflect thermal stability requirements for different environmental niches.

  • Crystal structures of PIPS from M. kansasii provide insights applicable to related enzymes in M. tuberculosis and M. bovis .

This comparative approach not only enhances our understanding of mycobacterial evolution but also identifies conserved features that represent potential broad-spectrum drug targets against multiple pathogenic mycobacteria.

How can isotope labeling techniques be applied to study the metabolic flux through pathways involving CDP-diacylglycerol pyrophosphatase in living mycobacterial cells?

Isotope labeling techniques provide powerful tools for investigating metabolic flux through pathways involving CDP-diacylglycerol pyrophosphatase in living mycobacterial cells:

• Selection of appropriate isotope tracers:

  • 13C-labeled glycerol or glucose: To track carbon flux into the glycerol backbone of phospholipids.

  • 13C-labeled fatty acids: To monitor incorporation into the acyl chains of CDP-DAG.

  • 14C-labeled cytidine: To specifically trace the CDP moiety through the pathway.

  • 32P or 33P isotopes: To follow phosphate incorporation and transfer.

• Experimental design considerations:

  • Pulse-chase experiments: Short pulse with labeled precursor followed by chase with unlabeled compound to track metabolic intermediates.

  • Steady-state labeling: Continuous exposure to labeled precursors until isotopic equilibrium is reached.

  • Time-course sampling: To capture dynamic changes in metabolite pools.

• Analytical techniques for metabolite detection and quantification:

  • Liquid chromatography-mass spectrometry (LC-MS/MS): For sensitive detection of labeled intermediates.

  • Nuclear magnetic resonance (NMR) spectroscopy: For detailed structural analysis of labeled compounds.

  • Thin-layer chromatography with radiometric detection: For straightforward visualization of 14C or 32P-labeled lipids.

• Data analysis and interpretation:

  • Isotopomer distribution analysis: To determine the pattern of label incorporation.

  • Metabolic flux modeling: Using computational approaches to calculate flux rates through different branches of the pathway.

  • Comparison between wild-type and genetically modified strains: To assess the impact of cdh activity modulation.

• Application to specific research questions:

  • Pathway bottleneck identification: Determining rate-limiting steps in phospholipid biosynthesis.

  • Drug effect studies: Measuring how inhibitors alter flux through the pathway.

  • Stress response analysis: Investigating metabolic rewiring under different environmental conditions.

These techniques allow researchers to move beyond static measurements of enzyme activity to understand the dynamic role of cdh in mycobacterial phospholipid metabolism under physiologically relevant conditions.