Recombinant Putative S-adenosyl-L-methionine-dependent methyltransferase MAP_4191c (MAP_4191c)

What is MAP_4191c and how does it function as a methyltransferase?

MAP_4191c is a putative S-adenosyl-L-methionine (AdoMet)-dependent methyltransferase from Mycobacterium avium paratuberculosis. Like other methyltransferases in this family, it likely catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to specific substrates, which could include proteins, DNA, RNA, or small molecules.

The enzyme's function can be experimentally determined through several approaches:

• Sequence alignment with characterized methyltransferases to identify conserved domains

• In vitro methylation assays using recombinant protein and potential substrates

• Gene knockout studies to observe phenotypic changes

• Complementation assays with known methyltransferase-deficient strains

Similar to PrmC in Chlamydia trachomatis, MAP_4191c likely contains the characteristic methyltransferase domain with AdoMet binding motifs . Functional analysis might reveal whether it methylates specific factors within Mycobacterium avium paratuberculosis, potentially playing roles in gene expression, protein function, or virulence.

What experimental systems can be established to study MAP_4191c activity?

To establish reliable experimental systems for studying MAP_4191c activity, researchers should consider:

• Heterologous expression systems:

  • E. coli-based expression using vectors like pQE-80L (similar to those used for chlamydial PrmC)

  • Mycobacterial expression systems for proper folding and post-translational modifications

  • Cell-free protein synthesis for rapid screening

• Complementation assays:

  • Similar to the approach used with C. trachomatis PrmC, where functionality was established by complementing E. coli K-12 prmC knockout strain (SC5)

  • Testing whether MAP_4191c can restore growth defects or functional deficiencies in methyltransferase-deficient strains

• Activity measurement systems:

  • Radioactive methyl transfer assays using [³H]-AdoMet

  • Fluorescence-based methylation detection systems

  • Mass spectrometry to identify methylated substrates

These systems should incorporate appropriate controls and variable manipulation as outlined in experimental design principles to ensure valid results .

How can the expression and purification of recombinant MAP_4191c be optimized?

Optimizing expression and purification of recombinant MAP_4191c requires systematic testing of multiple conditions:

Expression optimization:

• Test multiple expression vectors with different promoters (T7, tac, araBAD)

• Evaluate various E. coli strains (BL21(DE3), Rosetta, Arctic Express)

• Optimize induction parameters:

  • IPTG concentration (0.1-1.0 mM)

  • Temperature (16°C, 25°C, 37°C)

  • Induction time (4-24 hours)

• Consider codon optimization for E. coli if expression levels are low

Purification strategies:

• Test multiple affinity tags (His-tag, GST, MBP) for improved solubility

• Screen buffer conditions systematically:

  • pH range (6.0-8.5)

  • Salt concentration (100-500 mM NaCl)

  • Additives (glycerol, reducing agents)

• Implement multi-step purification:

  • Initial affinity chromatography

  • Ion exchange chromatography

  • Size exclusion chromatography for highest purity

Similar to experiments with PrmC, it's worth noting that even low levels of recombinant protein can be sufficient for functional complementation assays, as observed with chlamydial PrmC which was undetectable by SDS-PAGE but still provided functional complementation .

What are the most effective experimental designs for studying MAP_4191c substrate specificity?

Determining substrate specificity for MAP_4191c requires carefully designed experiments following robust experimental design principles:

• Hypothesis-driven approach:

  • Formulate clear null and alternate hypotheses about potential substrates

  • Design experiments that can specifically isolate the effect of MAP_4191c on target substrates

• Systematic variable manipulation:

  • Independent variables: potential substrates, reaction conditions, enzyme concentrations

  • Dependent variables: methylation levels, functional changes in substrates

  • Control variables: buffer conditions, temperature, co-factors

• Targeted substrate screening:

  • In vitro methylation assays with purified recombinant proteins, DNA, or RNA

  • Substrate competition assays to determine relative affinities

  • MS/MS analysis to identify methylation sites

• In vivo approaches:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling methods (BioID, APEX) to identify interacting partners

  • Comparative proteomics between wild-type and MAP_4191c knockout strains

This multi-faceted approach follows the experimental design steps of defining variables, writing hypotheses, and designing experimental treatments to isolate the specific activity of MAP_4191c .

How do experimental conditions affect MAP_4191c activity and what are the optimal assay parameters?

The activity of S-adenosyl-L-methionine-dependent methyltransferases like MAP_4191c can be significantly affected by experimental conditions. Researchers should systematically evaluate:

• Buffer optimization:

  Buffer Component	Range to Test	Typical Optimal Range
  pH	6.0-9.0	7.5-8.5
  NaCl	0-500 mM	50-150 mM
  MgCl₂	0-10 mM	1-5 mM
  DTT/β-ME	0-10 mM	1-5 mM
  Glycerol	0-20%	5-10%

• Reaction parameters:

  • Temperature effects (25°C, 30°C, 37°C, 42°C)

  • Incubation time (5 min to 24 hours)

  • Enzyme:substrate ratio (1:1 to 1:100)

  • AdoMet concentration (1-100 μM)

• Control experiments:

  • Heat-inactivated enzyme controls

  • S-adenosyl-L-homocysteine inhibition controls

  • AdoMet-free reactions

These parameters should be systematically tested using true experimental design with proper controls and randomization to establish valid cause-effect relationships . The activity measurements should follow the principle of varying one factor at a time while controlling others to isolate specific effects.

What approaches can detect post-translational modifications introduced by MAP_4191c?

Detecting post-translational methylation modifications introduced by MAP_4191c requires sensitive analytical techniques:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics: Digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact proteins

  • Multiple Reaction Monitoring (MRM) for targeted analysis

  • Electron Transfer Dissociation (ETD) for preserving labile modifications

• Antibody-based methods:

  • Western blotting with methylation-specific antibodies

  • Immunoprecipitation of methylated proteins

  • Immunofluorescence microscopy to determine subcellular localization

• Chemical labeling strategies:

  • Click chemistry approaches for detecting methylated residues

  • Methyl-SILAC for quantitative analysis of methylation

  • Bioorthogonal labeling of methylated substrates

• Functional assays:

  • Activity comparison between methylated and unmethylated proteins

  • Structural studies to determine methylation-induced conformational changes

Similar to studies with PrmC, which was shown to methylate release factors within the conserved GGQ motif , identifying the specific methylation sites on MAP_4191c substrates will provide crucial insights into its function and mechanism.

How can contradictory results in MAP_4191c functional studies be reconciled?

Contradictory results in functional studies of MAP_4191c or any S-adenosyl-L-methionine-dependent methyltransferase can stem from multiple factors. A systematic approach to reconcile such discrepancies includes:

• Experimental design analysis:

  • Review the experimental design for potential flaws in controlling variables

  • Assess if proper randomization and blinding techniques were employed

  • Evaluate whether the sample sizes provided adequate statistical power

• Methodological differences assessment:

  • Compare protein expression and purification protocols

  • Assess differences in assay conditions (pH, temperature, cofactors)

  • Evaluate substrate preparation methods and purity

• Strain and genetic background considerations:

  • Genetic differences between laboratory strains of M. avium paratuberculosis

  • Presence of compensatory mechanisms in different genetic backgrounds

  • Potential epigenetic effects influencing enzyme activity

• Technical approach to resolution:

  • Independent replication by neutral laboratories

  • Meta-analysis of existing data when multiple studies are available

  • Development of standardized protocols for the field

This approach follows proper experimental design principles by controlling extraneous variables, identifying confounding factors, and establishing reliable protocols to isolate the true effects of MAP_4191c .

What structural biology approaches can elucidate MAP_4191c function and substrate interactions?

Understanding the structural basis for MAP_4191c function requires multiple complementary approaches:

These approaches, when combined with functional data, can reveal critical information about the catalytic mechanism, substrate recognition, and potential for inhibitor development, similar to structural studies performed on other S-adenosyl-L-methionine-dependent methyltransferases.

How can novel substrates of MAP_4191c be systematically identified using proteome-wide approaches?

Identifying the complete substrate profile of MAP_4191c requires comprehensive screening approaches:

• Proteome-wide screening methods:

  • Protein microarray incubation with active MAP_4191c and labeled AdoMet

  • SILAC-based comparative proteomics between wild-type and knockout strains

  • Activity-based protein profiling using AdoMet analogs

  • Thermal proteome profiling to identify proteins stabilized by MAP_4191c interaction

• Genetic approaches:

  • Synthetic genetic array analysis to identify genetic interactions

  • CRISPR-Cas9 screens for genes with functional relationships to MAP_4191c

  • Transcriptome analysis of MAP_4191c knockout strains

• Biochemical enrichment strategies:

  • Affinity purification using catalytically inactive MAP_4191c as bait

  • Enrichment of methylated proteins/peptides using anti-methyl antibodies

  • Chemical capture of methylated residues followed by MS identification

• Bioinformatic prediction:

  • Machine learning approaches to predict methylation sites based on sequence context

  • Structural modeling to identify proteins with compatible binding interfaces

  • Evolutionary analysis to identify conserved potential substrates

This comprehensive approach draws on experimental design principles by systematically varying conditions and employing proper controls to establish valid substrate identification , while building upon methodologies used to study other methyltransferases like PrmC .

How does MAP_4191c activity contribute to Mycobacterium avium paratuberculosis pathogenesis?

The potential role of MAP_4191c in pathogenesis can be investigated through several experimental approaches:

• Virulence studies:

  • Generation of MAP_4191c knockout and complemented strains

  • Animal infection models comparing virulence of wild-type vs. mutant strains

  • Cell culture invasion and survival assays

  • Competitive infection assays to assess relative fitness

• Host-pathogen interaction studies:

  • Transcriptomics of host cells infected with wild-type vs. MAP_4191c mutants

  • Analysis of host immune response to different strains

  • Identification of host factors affected by MAP_4191c activity

• Regulatory network analysis:

  • Identification of genes differentially expressed in MAP_4191c mutants

  • ChIP-seq studies if MAP_4191c affects DNA-binding proteins

  • Proteomics to identify changes in protein expression or modification

This research direction parallels studies of other methyltransferases in bacterial pathogens, where alterations in methylation can significantly impact virulence, as observed with N6-adenine methylation in Salmonella, Yersinia pseudotuberculosis, and Vibrio cholerae .

What computational approaches can predict MAP_4191c function and interactions?

Advanced computational methods offer powerful tools for predicting MAP_4191c function:

• Structural bioinformatics:

  • Homology modeling based on known methyltransferase structures

  • Molecular dynamics simulations to study conformational dynamics

  • Virtual screening for potential substrates and inhibitors

  • QM/MM studies of catalytic mechanism

• Systems biology approaches:

  • Network analysis to place MAP_4191c in functional pathways

  • Genome-scale metabolic modeling to predict effects of MAP_4191c knockout

  • Flux balance analysis to understand metabolic impacts

• Machine learning applications:

  • Substrate prediction based on sequence and structural features

  • Development of specialized models trained on methyltransferase data

  • Integration of multi-omics data to predict functional relationships

• Evolutionary analysis:

  • Phylogenetic profiling to identify co-evolving proteins

  • Analysis of selection pressure on MAP_4191c sequence

  • Comparative genomics across mycobacterial species

These computational approaches should be validated through experimental testing following proper experimental design principles, including formulating testable hypotheses and designing controlled experiments to test predictions .

How can targeting MAP_4191c inform development of novel antimycobacterial strategies?

Exploring MAP_4191c as a potential therapeutic target involves several research directions:

• Target validation approaches:

  • Essentiality studies through conditional knockdown systems

  • Chemical genetics with small molecule inhibitors

  • Complementation studies with human methyltransferases to assess selectivity

• Inhibitor discovery strategies:

  Approach	Advantages	Challenges	Success Metrics
  High-throughput screening	Unbiased discovery	Resource intensive	Z-factor >0.5
  Fragment-based screening	Efficient chemical space exploration	Requires structural data	LE >0.3 kcal/mol per heavy atom
  Virtual screening	Cost-effective	Depends on model quality	Enrichment factor >10
  Rational design	Structure-guided	Limited to known scaffolds	IC₅₀ improvement over generations

• Mechanism-based inhibitor development:

  • AdoMet analogs as competitive inhibitors

  • Bisubstrate inhibitors linking AdoMet and substrate mimics

  • Allosteric inhibitors targeting regulatory sites

• Therapeutic potential assessment:

  • In vitro activity against M. avium paratuberculosis

  • Selectivity over human methyltransferases

  • Pharmacokinetic and toxicity evaluations

This research direction builds upon understanding of methyltransferase function in bacterial pathogens and applies rigorous experimental design principles to establish causal relationships between MAP_4191c inhibition and antimicrobial effects .