Recombinant Putative diacyglycerol O-acyltransferase Rv1425/MT1468 (Rv1425, MT1468)

Basic Research Questions

• What is Rv1425/MT1468 and what is its biochemical function?

  Rv1425 is a gene in Mycobacterium tuberculosis H37Rv, while MT1468 is the corresponding gene identifier in the CDC1551 strain. The protein is classified as a putative diacylglycerol O-acyltransferase belonging to the long-chain O-acyltransferase family .

  When expressed in E. coli, Rv1425 functions weakly as a triacylglycerol synthase, catalyzing the formation of triacylglycerol (TG) from diacylglycerol (DAG) and long-chain fatty acyl-CoA . The gene encodes a protein of 460 amino acids with a size of 1380 bp .

  The enzymatic reaction catalyzed is:

  Diacylglycerol + Acyl-CoA → Triacylglycerol + CoA

  Functional studies have shown that Rv1425 is related to the wax ester synthase/diacylglycerol acyltransferase (WSD) family of enzymes, with homologs found in other organisms such as Euglena gracilis (EgWSD3) .

• How does Rv1425/MT1468 compare structurally and functionally to other DGAT enzymes?

  Rv1425 differs significantly from the mammalian DGAT enzymes (DGAT1 and DGAT2). While mammalian DGAT1 and DGAT2 have divergent structures and functions, Rv1425 represents a bacterial type of WSD enzyme .

  Key differences include:

  Feature	Rv1425/MT1468	Mammalian DGAT1	Mammalian DGAT2
  Cellular location	Unknown, likely membrane-associated	Endoplasmic reticulum	ER and lipid droplet tethering
  Size	460 aa	Varies (~500 aa)	Varies (~400 aa)
  Activity level	Weak TG synthesis	High TG synthesis	High TG synthesis
  Structural features	Part of bacterial WSD family	Contains transmembrane domains	Contains lipid droplet targeting domain
  Role in pathogenesis	Unknown, potential role in M. tuberculosis lipid metabolism	Not applicable	Not applicable

  When studied alongside other WSD enzymes in expression systems, the Rv1425 homolog (EgWSD3) showed moderate activity compared to other WSD enzymes . Notably, in comparative studies in yeast, EgWSD3 demonstrated significant but impaired C28 accumulation compared to EgWSD2 and EgWSD5 .

• What expression systems and purification methods are optimal for recombinant Rv1425/MT1468?

  For recombinant expression of Rv1425/MT1468, prokaryotic expression systems are typically employed, with E. coli being the most common host .

  Methodological considerations for optimal expression include:

  1. Expression vector selection: Vectors with strong inducible promoters (like T7) are preferred for controlled expression.

  2. Fusion tags: N-terminal His-tags are commonly used to facilitate purification via nickel affinity chromatography .

  3. Expression conditions: Optimization of induction temperature (typically lower temperatures of 16-25°C), inducer concentration, and duration of expression to maximize soluble protein yield.

  4. Buffer formulation: PBS, pH 7.4, containing stabilizers such as trehalose (5%) has been successfully used for recombinant protein storage .

  5. Protein solubilization: Since Rv1425 likely contains transmembrane domains, detergent solubilization may be necessary to maintain protein in solution.

  Purification typically follows a multi-step approach:

  1. Affinity chromatography (Ni-NTA for His-tagged proteins)

  2. Size exclusion chromatography to remove aggregates

  3. Optional ion exchange chromatography for further purification

  Protein stability can be enhanced by including reducing agents like DTT (1mM) and cryoprotectants like trehalose (5%) in the storage buffer .

Advanced Research Questions

• What experimental design considerations are crucial when studying Rv1425/MT1468 enzymatic activity?

  When designing experiments to study Rv1425/MT1468 enzymatic activity, researchers should implement the following methodological approaches:

  1. Controls: Include both positive controls (known active DGAT enzymes) and negative controls (empty vector transformants, heat-inactivated enzyme) to validate experimental systems .

  2. Experimental design structure:

     • Consider a completely randomized design or randomized block design depending on variables being tested

     • Determine if a between-subjects or within-subjects design is most appropriate

     • Use appropriate sample sizes for statistical power

  3. Variables to consider:

     Variable Type	Examples for Rv1425 Studies
     Independent variables	Substrate concentrations, pH, temperature, cofactors
     Dependent variables	Enzyme activity rate, product formation
     Controlled variables	Buffer composition, protein concentration, incubation time
     Confounding variables	Protein stability, substrate accessibility, detergent effects

  4. Substrate specificity testing: Examine activity with various DAG species and acyl-CoA donors of different chain lengths and saturation .

  5. Analytical methods:

     • Thin-layer chromatography (TLC) for qualitative product identification

     • Gas chromatography-mass spectrometry (GC-MS) for quantitative analysis of lipid products

     • Radiolabeled substrate incorporation for sensitive detection of activity

  6. Data analysis approach:

     • Use appropriate statistical tests for hypothesis testing

     • Consider multivariate analysis for complex experimental designs

     • Present data in clear tables with appropriate statistical measures (means, standard deviations, p-values)

  Following these experimental design principles will help ensure reliable, reproducible results when characterizing the enzymatic properties of Rv1425/MT1468.

• How can researchers address and interpret contradictory experimental data regarding Rv1425/MT1468 function?

  When faced with contradictory experimental data regarding Rv1425/MT1468 function, researchers should employ a systematic approach to identify and resolve discrepancies:

  1. Identify sources of variability:

     • Expression systems (E. coli vs. yeast vs. mycobacterial hosts)

     • Assay conditions (pH, temperature, detergent concentration)

     • Protein construct design (full-length vs. truncated versions)

     • Detection methods (sensitivity differences between techniques)

  2. Methodological approaches to reconcile contradictions:

     • Comparative analysis: Directly compare methods using standardized protocols and identical protein preparations.

     • Multiple analytical techniques: Apply orthogonal methods to verify findings (e.g., combine TLC, GC-MS, and enzymatic assays).

     • Collaborative validation: Engage multiple laboratories to independently verify key findings using shared protocols and reagents.

     • Control for threats to internal validity: Address history effects, maturation, testing effects, instrumentation, selection bias, and other factors that may confound results .

  3. Systematic investigation of specific variables:

     Variable	Investigation Method
     Protein stability	Thermal shift assays, limited proteolysis to assess proper folding
     Substrate accessibility	Vary detergent types/concentrations, test liposome-incorporated enzyme
     Post-translational modifications	Compare enzyme from different expression systems, use mass spectrometry to identify modifications
     Cofactor requirements	Systematic screening of potential cofactors and activators

  4. Data interpretation framework:

     • Consider biological context (mycobacterial physiology vs. heterologous expression)

     • Evaluate physiological relevance of in vitro findings

     • Compare with related enzymes from the same family

     • Consider evolutionary conservation of function

  By systematically investigating contradictions using multiple approaches and carefully controlled experiments, researchers can develop a more comprehensive understanding of Rv1425/MT1468 function.

• What methods should be employed to accurately assess Rv1425/MT1468 enzymatic activity in vitro?

  Accurate assessment of Rv1425/MT1468 enzymatic activity requires specialized methods appropriate for membrane-associated acyltransferases:

  1. In vitro reconstitution systems:

     • Purified enzyme in detergent micelles

     • Enzyme incorporated into liposomes or nanodiscs

     • Mixed micelle systems with carefully controlled detergent/lipid ratios

  2. Substrate preparation:

     • DAG substrates prepared as mixed micelles or emulsions

     • Acyl-CoA donors in aqueous solution

     • Consideration of substrate presentation and accessibility

  3. Activity assay methods:

     Method	Protocol Overview	Advantages	Limitations
     Radioisotope-based assays	Use 14C-labeled acyl-CoA; extract and quantify labeled TG products	High sensitivity; direct measurement of product formation	Requires radioisotope handling; expensive
     Spectrophotometric coupled assays	Measure CoA release using coupled enzymes (e.g., DTNB reaction)	Continuous measurement; amenable to high-throughput	Potential for interference; indirect measurement
     LC-MS/MS quantification	Extract lipids and quantify DAG consumption and TG formation	Direct measurement of substrates and products; high specificity	Equipment intensive; complex sample preparation
     TLC separation with densitometry	Separate lipids by TLC and quantify spots	Visual verification of products; relatively simple	Semi-quantitative; lower sensitivity

  4. Enzyme kinetic analysis:

     • Determination of Km and Vmax for both DAG and acyl-CoA substrates

     • Evaluation of substrate specificity profiles

     • Assessment of potential inhibitors or activators

  5. Controls and validation:

     • Inactive enzyme controls (heat-inactivated or mutated catalytic residues)

     • Substrate-only and enzyme-only controls

     • Positive controls using well-characterized DGATs or WSDs

  6. Data analysis and presentation:

     • Plot enzyme kinetics using appropriate models (Michaelis-Menten, Lineweaver-Burk)

     • Present raw data alongside processed results

     • Include statistical analysis of replicate experiments

  These methodologies provide a comprehensive approach to accurately characterize the enzymatic activity of Rv1425/MT1468 in vitro.

• How might Rv1425/MT1468 contribute to Mycobacterium tuberculosis pathogenesis?

  The potential role of Rv1425/MT1468 in M. tuberculosis pathogenesis can be examined through several research perspectives:

  1. Lipid metabolism and energy storage:

     • M. tuberculosis is known to accumulate triacylglycerols during dormancy and stress conditions

     • Rv1425 may contribute to lipid body formation, providing energy reserves for long-term survival in host tissues

     • Triacylglycerol synthesis could be part of the adaptation to nutrient-limited environments in granulomas

  2. Host-pathogen interactions:

     • Modification of host cell lipid metabolism during infection

     • Potential role in altering macrophage function through manipulation of lipid signaling

     • Possible involvement in forming the complex cell wall lipids that contribute to M. tuberculosis virulence

  3. Experimental approaches to investigate pathogenic roles:

     Approach	Methodology	Expected Insights
     Gene knockout studies	Create Rv1425 deletion mutants using specialized transposon systems	Determine if Rv1425 is essential for growth or virulence in various conditions
     Conditional expression	Regulate Rv1425 expression using inducible promoters	Assess effects of varying expression levels on bacterial phenotype
     Transcriptional profiling	RNA-seq analysis under various stress conditions	Determine conditions that regulate Rv1425 expression
     Infection models	Compare wild-type and Rv1425-mutant strains in cellular and animal models	Assess impact on colonization, persistence, and pathogenicity

  4. Gene expression data insights:

     Based on available transcriptomic data (search result ), Rv1425 expression shows specific patterns that may correlate with pathogenic phases:

     Condition	Expression Level	Implication
     Standard growth	Moderate expression	Basal metabolic function
     Nutrient starvation	Potentially upregulated (based on related genes)	Role in adaptation to host environment
     Dormancy	Expression changes may correlate with lipid accumulation	Potential role in persistent infection

  5. Structure-function relationship in pathogenesis:

     • The enzymatic activity contributing to triacylglycerol synthesis may be directly linked to persistence mechanisms

     • Comparison with other bacterial pathogens that utilize lipid metabolism for virulence

     • Evaluation as a potential drug target based on essentiality or contribution to virulence

  Understanding the role of Rv1425 in M. tuberculosis pathogenesis requires integrating data from biochemical, genetic, and infection model studies to establish its contribution to the complex lifecycle of this pathogen.

• What site-directed mutagenesis strategies can be employed to investigate Rv1425/MT1468 catalytic mechanism?

  Site-directed mutagenesis provides a powerful approach to investigate the catalytic mechanism of Rv1425/MT1468. A comprehensive strategy should include:

  1. Identification of catalytic and functional residues:

     • Sequence alignment with characterized DGAT/WSD enzymes to identify conserved residues

     • Structural prediction tools to identify potential active site residues

     • Homology modeling based on related enzymes with known structures

  2. Systematic mutagenesis approach:

     Residue Type	Mutagenesis Strategy	Rationale
     Predicted catalytic histidine	H→A, H→N mutations	Test role in acyl transfer reaction and hydrogen bonding
     Conserved serine/threonine	S/T→A mutations	Assess importance in substrate binding or catalysis
     Hydrophobic residues in putative substrate-binding regions	Conservative (I→L) and non-conservative (I→A) mutations	Determine role in substrate specificity
     Charged residues	Charge reversal (D→K, K→E)	Evaluate electrostatic contributions to activity
     Cysteine residues	C→S mutations	Investigate potential regulatory or structural roles

  3. Expression and purification of mutants:

     • Use standardized expression systems (E. coli) for consistent comparison

     • His-tag or other affinity tags for purification

     • Verify protein folding and stability for each mutant using circular dichroism or thermal shift assays

  4. Enzymatic activity characterization:

     • Compare wild-type and mutant activity using standardized assays

     • Determine kinetic parameters (Km, Vmax) for each mutant

     • Assess substrate specificity changes resulting from mutations

  5. Structural analysis of mutations:

     • Circular dichroism to assess secondary structure changes

     • Intrinsic fluorescence to monitor tertiary structure alterations

     • Thermal stability analysis to determine if mutations affect protein stability

     • Computational modeling to interpret experimental findings

  6. Data interpretation framework:

     • Categorize mutations based on effects (complete loss of activity, reduced activity, altered specificity)

     • Develop a model of the catalytic mechanism based on mutational effects

     • Compare findings with mechanisms proposed for related enzymes

     • Iterate with additional mutations to refine the mechanistic model

  This comprehensive mutagenesis approach will provide detailed insights into the catalytic mechanism of Rv1425/MT1468 and its relationship to enzyme structure.

• How can researchers develop robust experimental controls when studying Rv1425/MT1468?

  Developing robust experimental controls is essential for obtaining reliable and interpretable data when studying Rv1425/MT1468. A comprehensive control strategy should include:

  1. Genetic and expression controls:

     • Empty vector controls processed identically to Rv1425-expressing constructs

     • Expression of known active and inactive DGAT enzymes as positive and negative controls

     • Expression of Rv1425 with site-directed mutations in predicted catalytic residues as functional negative controls

  2. Enzymatic activity controls:

     Control Type	Implementation	Purpose
     Heat-inactivated enzyme	Boil purified Rv1425 for 10 minutes	Control for non-enzymatic reactions
     Substrate-only controls	Reaction mixtures without enzyme	Monitor spontaneous substrate degradation
     CoA release controls	Reactions without DAG substrate	Check for non-specific acyl-CoA hydrolysis
     Known DGAT enzyme	Include purified DGAT1 or DGAT2	Benchmark for activity comparison
     Chemical inhibitor	Include known DGAT inhibitors	Verify specific inhibition patterns

  3. Controls for experimental validity threats:

     • Randomization of sample processing to minimize systematic errors

     • Blinding of sample identity during analysis when possible

     • Temporal controls to account for potential time-dependent variations

     • Technical and biological replicates to assess reproducibility and variation

  4. Addressing specific threats to internal validity:
     Based on search result , researchers should control for:

     Validity Threat	Control Strategy
     History effects	Include time-matched controls for all experimental conditions
     Maturation	Use appropriate time series controls if processes change over time
     Testing effects	Consider how preliminary measurements might affect subsequent results
     Instrumentation	Calibrate equipment regularly and include standard curves
     Selection bias	Use random assignment and appropriate statistical blocking
     Statistical regression	Account for extreme measurements and natural variation

  5. Data analysis controls:

     • Use appropriate statistical tests with controls for multiple comparisons

     • Include normalization controls for inter-experimental comparisons

     • Implement appropriate transformations for non-normally distributed data

     • Use internal standards for quantitative analyses

  6. Reporting standards:

     • Document all control experiments in methods sections

     • Present control data alongside experimental results when appropriate

     • Address limitations and potential confounding factors in discussion

     • Follow field-specific reporting guidelines for enzyme characterization studies

  By implementing this comprehensive control strategy, researchers can maximize confidence in their findings regarding Rv1425/MT1468 function and mechanism.

• What analytical methods should be employed to accurately quantify Rv1425/MT1468 enzymatic products?

  Accurate quantification of Rv1425/MT1468 enzymatic products requires sophisticated analytical methods appropriate for lipid analysis:

  1. Chromatographic separation techniques:

     • Thin-layer chromatography (TLC) for initial separation and visualization

     • High-performance liquid chromatography (HPLC) for improved resolution

     • Gas chromatography (GC) for analysis of fatty acid composition

  2. Mass spectrometry-based approaches:

     • LC-MS/MS for sensitive detection and quantification of intact lipids

     • GC-MS for analysis of fatty acid methyl esters derived from product lipids

     • MALDI-TOF MS for rapid screening of lipid profiles

  3. Quantification methods comparison:

     Method	Description	Sensitivity	Specificity	Workflow Complexity
     TLC with densitometry	Separate lipids on silica plates; quantify by densitometric scanning	Moderate	Moderate	Low
     HPLC with ELSD/CAD	Separate lipids by HPLC; detect using evaporative light scattering or charged aerosol detection	High	Moderate	Moderate
     LC-MS/MS	Separate by HPLC; identify and quantify by tandem MS	Very high	Very high	High
     Radioisotope detection	Use 14C-labeled substrates; quantify products by scintillation counting	Very high	Moderate	Moderate
     Enzyme-coupled assays	Detect released CoA using coupled enzymatic reactions	Moderate	Low	Low

  4. Sample preparation protocols:

     • Total lipid extraction using Bligh-Dyer or Folch methods

     • Solid-phase extraction for lipid class separation

     • Derivatization strategies for improved detection sensitivity

     • Internal standards for quantitative accuracy

  5. Data analysis considerations:

     • Calibration curves using authentic standards for absolute quantification

     • Internal standards to correct for extraction and ionization efficiency

     • Statistical analysis of technical and biological replicates

     • Method validation parameters (linearity, accuracy, precision, limits of detection)

  6. Method selection guidance:

     • For initial activity screening: TLC or enzyme-coupled assays

     • For detailed characterization: LC-MS/MS or GC-MS

     • For kinetic studies: Continuous assays (enzyme-coupled) or time-point sampling

     • For substrate specificity: MS-based methods with detailed lipid identification

  These analytical approaches provide a comprehensive toolkit for accurately quantifying the enzymatic products of Rv1425/MT1468, enabling detailed characterization of its biochemical function.