Recombinant Uncharacterized oxidoreductase Rv0945/MT0971 (Rv0945, MT0971)

What is the functional annotation of Rv0945/MT0971?

Rv0945, also designated as MT0971 in some strain annotations, is annotated as a ketoacyl reductase belonging to the oxidoreductase family in Mycobacterium tuberculosis . This protein is part of the M. tuberculosis genome (strain H37Rv) and appears to be involved in redox reactions that may contribute to mycobacterial metabolism. While its precise function remains incompletely characterized, sequence analysis suggests its role in fatty acid metabolism, potentially participating in the mycolic acid biosynthesis pathway that is critical for cell wall formation in mycobacteria.

How can I determine if Rv0945 is essential for M. tuberculosis survival?

To determine the essentiality of Rv0945, researchers typically employ several complementary approaches:

• Gene knockout studies: Generate a deletion mutant of Rv0945 using homologous recombination or CRISPR-Cas9 systems, followed by assessment of viability under standard growth conditions.

• Transposon mutagenesis: Analyze global transposon insertion libraries to identify whether Rv0945 can tolerate insertions without losing viability.

• Conditional expression systems: Create strains where Rv0945 expression is controlled by inducible promoters to observe effects of protein depletion on growth and survival.

• Comparative genomics: Examine conservation across mycobacterial species and strains to infer selective pressure for retention.

Expression data shows that MT0971 (Rv0945) has variable expression levels across different experimental conditions, with numerical values showing expression ratios that suggest potential regulation under specific conditions .

What is the expression profile of Rv0945 under different growth conditions?

Based on available data, the expression profile of Rv0945/MT0971 varies considerably across different conditions. Expression data indicates MT0971 has values of 98.21 and 92.12 in two experimental conditions compared to 91.20 and 87.74 in others, with fold-change ratios of approximately 0.94, suggesting relatively stable expression across some tested conditions .

For comprehensive expression profiling, researchers should:

• Perform quantitative RT-PCR analysis under various growth conditions (aerobic, anaerobic, nutrient limitation, different carbon sources)

• Conduct RNA-seq experiments in vitro and ex vivo (e.g., macrophage infection models)

• Use reporter constructs (such as GFP fusions) to monitor expression in real-time during infection

• Compare expression during different growth phases and stress conditions relevant to tuberculosis pathogenesis

What are the optimal conditions for recombinant expression of Rv0945?

The optimal conditions for recombinant expression of Rv0945 would likely follow protocols similar to those used for other M. tuberculosis oxidoreductases. Based on methodologies applied to similar proteins:

• Expression system selection:

  • E. coli BL21(DE3) or Rosetta strains typically provide good expression for mycobacterial proteins

  • Consider codon optimization for E. coli expression, as mycobacterial genes often have different codon usage patterns

• Vector and tag selection:

  • pET series vectors with N-terminal 6xHis tag facilitate purification

  • For improved solubility, consider fusion tags such as MBP, SUMO, or Thioredoxin

• Induction conditions:

  • IPTG concentration: 0.1-0.5 mM typically provides balance between expression and solubility

  • Temperature: Lower temperatures (16-20°C) often improve folding of mycobacterial proteins

  • Duration: Extended expression (16-20 hours) at lower temperatures may increase yield of soluble protein

• Lysis buffer optimization:

  • Include glycerol (10-15%) to stabilize protein structure

  • Add reducing agents (5-10 mM β-mercaptoethanol or 1-2 mM DTT) to maintain thiol groups

  • Consider detergents (0.1% Triton X-100) if membrane association is suspected

Similar oxidoreductases from M. tuberculosis have been successfully expressed using these approaches, though protein-specific optimization will likely be necessary .

How can reductive methylation improve crystallization of Rv0945?

Reductive methylation can significantly improve crystallization properties of mycobacterial proteins that initially yield poor-quality crystals. Drawing from experience with Rv0765c (another oxidoreductase from M. tuberculosis):

• Methylation protocol:

  • Treat purified protein with dimethylamine-borane complex and formaldehyde

  • Perform reaction at 4°C in buffer containing 50 mM HEPES pH 7.5, 250 mM NaCl

  • Quench with addition of glycine and purify by size-exclusion chromatography

• Expected outcomes:

  • Methylation modifies surface lysine residues, reducing surface entropy

  • This modification can alter crystal packing interactions and improve diffraction quality

  • In the case of Rv0765c, methylation transformed crystals from diffracting to only 7Å resolution to a new crystal form diffracting to 3.2Å resolution

• Verification steps:

  • Confirm methylation via MALDI-TOF mass spectrometry

  • Verify that enzymatic activity is retained after modification

  • Compare crystallization behavior of native and methylated protein

• Optimization considerations:

  • Screen crystallization conditions specifically designed for methylated proteins

  • Test additives such as n-butanol or acetonitrile, which improved crystal quality for Rv0765c

  • Consider seeding techniques to improve crystal size and quality

The dramatic improvement observed for Rv0765c (from 7Å to 3.2Å resolution) suggests this approach could be valuable for Rv0945 crystallization efforts .

What is the recommended purification workflow for obtaining highly pure Rv0945?

For purifying recombinant Rv0945 to homogeneity suitable for structural and functional studies, the following workflow is recommended:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Wash with increasing imidazole concentrations (20-50 mM) to remove non-specific binding

  • Elute with 250-300 mM imidazole buffer

• Intermediate purification:

  • Ion exchange chromatography (predicted pI will determine whether cation or anion exchange is appropriate)

  • Consider tag removal using appropriate protease if tag might interfere with function

  • Verify initial purity by SDS-PAGE before proceeding

• Polishing step:

  • Size-exclusion chromatography using Superdex 75 or 200 columns

  • Buffer conditions: 50 mM Tris or HEPES pH 7.5-8.0, 150-300 mM NaCl, 5% glycerol, 1 mM DTT

  • Analyze oligomeric state and homogeneity by comparing elution volume to standards

• Quality control:

  • Assess final purity by SDS-PAGE (>95% purity recommended)

  • Verify identity by mass spectrometry

  • Perform dynamic light scattering to confirm monodispersity

  • Test enzyme activity with appropriate substrate to confirm functionality

Similar approaches have been successfully applied to other M. tuberculosis oxidoreductases, including Rv0765c .

What crystallization conditions should be explored first for Rv0945?

Based on successful crystallization of similar M. tuberculosis oxidoreductases, the following crystallization strategies are recommended for Rv0945:

• Initial screening:

  • Commercial sparse matrix screens (Hampton Research Crystal Screens, Molecular Dimensions JCSG+)

  • Protein concentration range: 5-15 mg/ml

  • Temperature: Both 4°C and 20°C setups

  • Methods: Vapor diffusion (sitting and hanging drop) with 1:1 and 2:1 protein:reservoir ratios

• Conditions to prioritize based on success with similar proteins:

  • PEG-based conditions (PEG 3350, 4000, 8000 at 10-25%)

  • Salt conditions including tartrate salts (sodium/potassium tartrate 0.8-1.2 M)

  • Buffer systems at pH 7.0-8.5 (HEPES, Tris)

  • Additives: n-butanol (0.5-1.0%) or acetonitrile (3-5%) which improved diffraction for Rv0765c

• Optimization strategies:

  • Fine gradient screens around initial hits

  • Additive screens for promising conditions

  • Seeding techniques (microseed matrix screening)

  • If initial crystals diffract poorly, pursue reductive methylation as performed for Rv0765c

• Alternative approaches if vapor diffusion fails:

  • Microbatch under oil

  • Lipid cubic phase for proteins with hydrophobic regions

  • Counter-diffusion in capillaries

The experience with Rv0765c suggests that crystallization conditions containing tartrate salts with HEPES buffer (pH 7.5-8.0) supplemented with small organic additives may be particularly promising .

How can homology modeling guide research on Rv0945 before crystal structure determination?

Homology modeling can provide valuable structural insights to guide experimental work with Rv0945 before the crystal structure is determined:

• Template selection:

  • Identify proteins with known structures and sequence homology to Rv0945

  • Potential templates include other mycobacterial oxidoreductases and ketoacyl reductases

  • Based on annotation as a ketoacyl reductase, structures of related enzymes from the short-chain dehydrogenase/reductase family would be appropriate templates

• Model building and validation:

  • Generate multiple models using software such as SWISS-MODEL, Phyre2, or MODELLER

  • Evaluate models using PROCHECK, VERIFY3D, and ProSA

  • Select the model with the best validation scores for further analysis

• Structure-guided experimental design:

  • Identify putative active site residues for site-directed mutagenesis

  • Predict substrate binding regions to guide docking studies

  • Map sequence conservation onto structural models to identify functionally important regions

  • Design truncation constructs based on domain predictions to improve expression or crystallization

• Model refinement steps:

  • Molecular dynamics simulations to assess stability of the model

  • Refinement of loop regions that may differ from template structures

  • Integration of experimental data (if available) to constrain the model

The experience with Rv0765c shows that structural homology with characterized oxidoreductases can provide valuable insights into protein function and guide experimental approaches .

What approaches can determine the oligomeric state of Rv0945 in solution?

Determining the accurate oligomeric state of Rv0945 in solution is critical for functional and structural studies. Several complementary techniques should be employed:

• Size-exclusion chromatography (SEC):

  • Compare elution volume with known molecular weight standards

  • Use multi-angle light scattering (SEC-MALS) for absolute molecular weight determination

  • Analyze concentration-dependent changes in elution profile to detect dynamic oligomerization

• Analytical ultracentrifugation (AUC):

  • Sedimentation velocity experiments to determine sedimentation coefficient

  • Sedimentation equilibrium to determine absolute molecular weight and association constants

  • Model fitting to determine stoichiometry of multi-component systems

• Native mass spectrometry:

  • Direct measurement of intact protein complexes

  • Can distinguish between different oligomeric species in solution

  • Provides information about non-covalent interactions

• Small-angle X-ray scattering (SAXS):

  • Generate low-resolution envelope of protein in solution

  • Compare experimental scattering with theoretical scattering of models

  • Derive parameters like radius of gyration and maximum particle dimension

• Chemical crosslinking:

  • Use bifunctional reagents like glutaraldehyde or BS3

  • Analyze products by SDS-PAGE and mass spectrometry

  • Identify interfaces through crosslinked peptide analysis

Many oxidoreductases from the short-chain dehydrogenase/reductase family function as dimers or tetramers, so these oligomeric states should be specifically evaluated for Rv0945.

What assays can be used to determine the substrate specificity of Rv0945?

As an annotated ketoacyl reductase, determining the substrate specificity of Rv0945 requires systematic screening approaches:

• Spectrophotometric assays:

  • Monitor NAD(P)H oxidation or NAD(P)+ reduction at 340 nm

  • Screen various fatty acyl substrates with different chain lengths (C4-C24)

  • Test both straight-chain and branched-chain substrates

  • Compare kinetic parameters (kcat, KM) across substrate series

• High-throughput substrate screening:

  • Prepare substrate libraries of potential ketoacyl compounds

  • Use microplate-based assays to screen activity across many substrates

  • Follow up on hits with detailed kinetic characterization

• Coupled enzyme assays:

  • Link Rv0945 activity to auxiliary enzymes producing measurable signals

  • Use biosensors that detect product formation

  • Consider redox-sensitive fluorescent probes to detect activity

• Mass spectrometry-based assays:

  • Direct detection of substrate consumption and product formation

  • Untargeted metabolomics approaches to identify novel substrates

  • Isotope labeling to track reaction progression and mechanism

• Activity-based protein profiling:

  • Use mechanism-based inhibitors or substrate analogs

  • Identify active site residues through labeling experiments

  • Compare with other characterized ketoacyl reductases

The systematic characterization of substrate preference will provide insights into the biological role of Rv0945 in mycobacterial metabolism.

How can I identify potential physiological partners of Rv0945 in mycobacterial metabolism?

Identifying the physiological partners and metabolic context of Rv0945 requires multiple complementary approaches:

• Genomic context analysis:

  • Examine gene neighborhood of Rv0945 in M. tuberculosis genome

  • Look for co-occurrence patterns across mycobacterial species

  • Identify potential operon structures or functionally related genes

• Protein-protein interaction methods:

  • Pull-down assays using tagged Rv0945 as bait

  • Bacterial two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches (BioID or APEX)

• Metabolic pathway analysis:

  • Based on the ketoacyl reductase annotation, map Rv0945 to fatty acid or polyketide synthesis pathways

  • Perform metabolomics analysis comparing wild-type and Rv0945 mutant strains

  • Use stable isotope labeling to track metabolic flux through pathways involving Rv0945

• Expression correlation analysis:

  • Analyze transcriptomic datasets to identify genes co-expressed with Rv0945

  • Focus on conditions where Rv0945 shows significant expression changes

  • The expression data from search result shows MT0971/Rv0945 has specific expression patterns that may correlate with other genes

A table presenting example expression data for Rv0945/MT0971 compared to other M. tuberculosis genes shows:

This expression pattern can be compared with other genes to identify potential functional relationships .

What approaches can determine if Rv0945 is involved in M. tuberculosis virulence?

To establish whether Rv0945 contributes to M. tuberculosis virulence and pathogenesis:

• Gene knockout and complementation studies:

  • Generate Rv0945 deletion mutant

  • Create complemented strain expressing wild-type Rv0945

  • Create point mutants targeting predicted catalytic residues

• Infection models:

  • Compare wild-type, knockout, and complemented strains in:

    • Macrophage infection assays (survival, replication, immunomodulation)

    • Mouse infection models (bacterial burden, histopathology, survival)

    • Human cell line infection models

  • Measure both bacterial fitness and host response parameters

• Gene expression analysis during infection:

  • Monitor Rv0945 expression during different stages of infection

  • Compare expression in active vs. latent infection models

  • Correlate expression with specific host microenvironments

• Specific virulence-related phenotypes:

  • Resistance to host-derived stresses (oxidative, nitrosative, acidic)

  • Cell wall integrity and composition

  • Persistence under antibiotic pressure

  • Biofilm formation capacity

• Comparative genomics across clinical isolates:

  • Analyze sequence variation in Rv0945 across clinical strains

  • Correlate specific variants with virulence phenotypes

  • Examine selection pressure on Rv0945 in evolving populations

These approaches will establish whether Rv0945 plays a direct or indirect role in the pathogenesis of M. tuberculosis.

How can I develop specific inhibitors targeting Rv0945?

Developing specific inhibitors against Rv0945 requires a rational drug design approach:

• Structure-based inhibitor design:

  • Use crystal structure or homology model of Rv0945

  • Identify and characterize the active site pocket

  • Perform virtual screening of compound libraries

  • Design compounds that interact with catalytic residues

• High-throughput screening approaches:

  • Develop a reliable enzymatic assay amenable to HTS format

  • Screen diverse chemical libraries (10,000-100,000 compounds)

  • Establish clear criteria for hit identification (e.g., >50% inhibition at 10 μM)

  • Confirm hits with dose-response curves and secondary assays

• Fragment-based drug discovery:

  • Screen libraries of low molecular weight compounds (fragments)

  • Use NMR, thermal shift assays, or crystallography to detect binding

  • Link or grow fragments to increase potency and specificity

  • Optimize physicochemical properties for mycobacterial penetration

• Validation of inhibitors:

  • Determine mechanism of inhibition (competitive, non-competitive)

  • Measure activity against purified enzyme and whole cells

  • Assess specificity against human homologs

  • Evaluate cytotoxicity in mammalian cells

• Structure-activity relationship studies:

  • Synthesize analogs of lead compounds

  • Correlate structural features with inhibitory potency

  • Optimize for mycobacterial penetration and target engagement

The validation of Rv0945 as a potential drug target would require demonstrating its essentiality or significant contribution to virulence as discussed in previous sections.

What are the challenges in determining the redox potential of Rv0945 and its impact on catalytic activity?

Determining the redox potential of Rv0945 and understanding its relationship to catalytic function presents several technical challenges:

• Methodology for redox potential determination:

  • Cyclic voltammetry requires electrode modification for protein immobilization

  • Spectroelectrochemical methods need specific equipment setups

  • Redox-sensitive dyes must not interfere with protein function

  • Potential reference points must be carefully calibrated

• Protein preparation considerations:

  • Maintain native conformation during measurements

  • Control oxidation state during purification and storage

  • Ensure homogeneity of the sample (single redox state)

  • Account for effects of buffer components and pH

• Correlation with catalytic properties:

  • Measure enzyme activity under defined redox conditions

  • Establish whether redox state affects substrate binding or catalytic rate

  • Determine if protein undergoes conformational changes with redox state

  • Identify key residues involved in redox sensing

• Physiological relevance assessment:

  • Compare in vitro measurements with estimated in vivo redox environment

  • Consider compartmentalization effects in the bacterial cell

  • Evaluate redox cycling during catalytic turnover

  • Assess impact of host-derived oxidative stress on function

• Experimental design recommendations:

  • Use anaerobic chambers to control oxygen exposure

  • Include redox buffers to maintain defined redox potential

  • Consider protein engineering to introduce spectroscopic probes

  • Compare wild-type and mutant proteins with altered redox properties

These approaches will provide insights into how the redox properties of Rv0945 influence its catalytic mechanism and potential regulation in the cellular environment.

How can cryo-EM be applied to study the structural dynamics of Rv0945 in complex with its substrates?

Cryo-electron microscopy (cryo-EM) offers unique advantages for studying Rv0945 structural dynamics:

• Sample preparation strategies:

  • Prepare Rv0945 at 1-5 mg/ml in buffer optimized for grid preparation

  • Capture multiple functional states by incubating with:

    • Substrates at various concentrations

    • Product analogs

    • Cofactors (NAD(P)H/NAD(P)+)

    • Inhibitors or transition state analogs

  • Apply sample to glow-discharged grids with thin carbon support

  • Optimize blotting conditions to achieve thin, uniform ice

• Data collection parameters:

  • Collect on high-end microscope (300 kV, direct electron detector)

  • Use movie mode with frame alignment to correct beam-induced motion

  • Implement dose weighting to minimize radiation damage

  • Collect at high defocus range for small proteins (~30 kDa)

• Image processing workflow:

  • Use reference-free 2D classification to select homogeneous particles

  • Perform ab initio reconstruction to generate initial models

  • Apply 3D classification to identify conformational heterogeneity

  • Refine structures to highest possible resolution

• Analysis of conformational dynamics:

  • Compare substrate-bound and apo states

  • Map conformational changes in active site upon substrate binding

  • Identify large-scale domain movements during catalytic cycle

  • Visualize oligomerization interfaces and their dynamics

• Integration with other structural methods:

  • Combine with X-ray crystallography for atomic details of active site

  • Validate models with cross-linking mass spectrometry

  • Correlate structural findings with kinetic data

  • Use molecular dynamics simulations to interpret conformational ensembles

Cryo-EM is particularly valuable for capturing the conformational landscape of Rv0945 under near-physiological conditions without crystal packing constraints.

How does Rv0945 expression change during different phases of M. tuberculosis infection?

Understanding the expression dynamics of Rv0945 throughout the infection cycle provides insights into its functional importance:

• Expression analysis methods:

  • Quantitative RT-PCR from infected tissue samples

  • RNA-seq of bacteria isolated from various infection models

  • Reporter strains (GFP/luciferase fusions) for real-time monitoring

  • Proteomics analysis of bacterial proteins during infection

• Key infection phases to analyze:

  • Early infection (initial macrophage entry)

  • Active replication phase

  • Granuloma formation

  • Latent/dormant state

  • Reactivation

• Correlation with environmental conditions:

  • Hypoxia (oxygen-limited conditions)

  • Nutrient limitation

  • Acidic pH

  • Exposure to reactive oxygen/nitrogen species

  • Exposure to host lipids

• Comparison with other metabolic genes:

  • Analyze co-expression patterns with other fatty acid metabolism genes

  • Compare with expression of known virulence factors

  • Limited data suggests MT0971/Rv0945 shows specific expression patterns that may correlate with infection stages, with expression values of 98.21, 92.12, 91.20, and 87.74 under different conditions

Understanding these expression patterns will help determine when Rv0945 activity is most critical during infection and identify potential intervention points.

What methodologies can determine if Rv0945 contributes to antibiotic resistance in M. tuberculosis?

To investigate potential connections between Rv0945 and antibiotic resistance:

• Genetic approaches:

  • Generate Rv0945 overexpression strains

  • Create knockout or knockdown mutants

  • Determine minimum inhibitory concentrations (MICs) for various antibiotics

  • Perform antibiotic killing curve analysis

• Evolution experiments:

  • Subject wild-type and Rv0945 mutant strains to increasing antibiotic concentrations

  • Sequence evolved strains to identify compensatory mutations

  • Analyze frequency of Rv0945 mutations in clinically resistant isolates

  • Perform allelic exchange to validate contributions of specific mutations

• Biochemical mechanisms investigation:

  • Test if Rv0945 directly modifies antibiotics (enzymatic inactivation)

  • Determine if Rv0945 affects cell wall permeability

  • Assess impacts on efflux pump expression or activity

  • Measure changes in redox homeostasis affecting antibiotic activity

• Systems biology approaches:

  • Perform transcriptomics/proteomics on Rv0945 mutants with/without antibiotics

  • Identify metabolic changes that might contribute to persistence

  • Map Rv0945 into known resistance networks

  • Model flux changes in pathways affected by Rv0945 activity

• Practical assay development:

  • Design high-throughput screening for compounds that synergize with antibiotics by targeting Rv0945

  • Develop biomarkers for Rv0945-mediated resistance mechanisms

  • Create diagnostic tools to identify resistance patterns involving Rv0945

These approaches will determine whether Rv0945 directly or indirectly contributes to antibiotic resistance phenotypes in M. tuberculosis.