Recombinant Bacteroides thetaiotaomicron tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG (mnmG), partial

Basic Research Questions

• What is the MnmG enzyme and what is its function in Bacteroides thetaiotaomicron?

  MnmG is an evolutionarily conserved bacterial protein that, together with MnmE, forms the MnmEG complex responsible for modifying transfer RNAs (tRNAs) that decode NNA/NNG codons. Specifically, in B. thetaiotaomicron and other bacteria, this complex catalyzes the addition of a 5-carboxymethylaminomethyl (cmnm^5) or 5-taurinomethyl (τm^5) group to position 5 of the anticodon wobble uridine (U34) of several tRNA species . While MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH2THF), MnmG binds flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH). This enzymatic complex is critical for proper codon recognition and translation fidelity .

• How does the MnmEG complex in B. thetaiotaomicron compare to the complex in E. coli?

  The MnmEG complex in both organisms shares fundamental mechanistic similarities. In E. coli, the complex modifies tRNAs decoding NNA/NNG codons, catalyzing reactions that add either an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine . The E. coli pathway involves an additional enzyme, the bi-functional MnmC, which contains two domains: MnmC(o) transforms cmnm^5 into nm^5, and MnmC(m) converts nm^5 into mnm^5 (5-methylaminomethyl) . Similar biochemical pathways likely exist in B. thetaiotaomicron, though specific differences in substrate specificity or regulation may be present based on the metabolic adaptations of this gut-resident bacterium .

• What cofactors are required for MnmG activity?

  MnmG activity requires several cofactors. The MnmG protein binds flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH), which are crucial for its enzymatic function . The complete MnmEG complex also requires guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH2THF), which bind to the MnmE component. Additionally, glycine serves as a substrate for the complex when catalyzing the installation of the cmnm^5 group, in a reaction that requires GTP hydrolysis . Alternative nucleophiles such as taurine and ammonia can also be utilized by the complex to form different modified uridines .

Advanced Research Questions

• What is the role of flavin-iminium FADH[N5=CH2]+ in the MnmEG reaction mechanism?

  Flavin-iminium FADH[N5=CH2]+ serves as a central intermediate in the MnmEG reaction mechanism. Biochemical evidence indicates that this intermediate is essential for transferring the methylene group from methylenetetrahydrofolate (CH2THF) to the C5 position of the wobble uridine (U34) in target tRNAs . Experiments using a synthetic FADH[N5=CD2]+ analogue have unambiguously demonstrated the intermediacy of FAD in this methylene transfer process. Furthermore, when MnmEG reactions containing the deuterated flavin-iminium intermediate were conducted with alternate nucleophiles like taurine and ammonia, the anticipated U34-modified tRNAs were formed. This confirms that FAD[N5=CH2]+ acts as a universal intermediate for all MnmEG homologues, regardless of the specific nucleophile used in the reaction .

• How does the expression of MnmG correlate with oxidative stress responses in B. thetaiotaomicron?

  While direct evidence linking MnmG expression to oxidative stress in B. thetaiotaomicron is limited in the provided search results, the organism does exhibit complex responses to oxidative environments that may involve translational regulation. B. thetaiotaomicron is known to be highly susceptible to oxidative environments . When metabolizing rhamnose rather than glucose, B. thetaiotaomicron demonstrates improved resistance to oxidative stress, with reduced reactive oxygen species (ROS) production . This metabolic adaptation involves the regulation of specific genes by the rhamnose metabolism regulator RhaR. While the search results don't explicitly connect MnmG to this pathway, proper tRNA modification by enzymes like MnmG is critical for effective translation, which could play a role in the expression of oxidative stress response proteins. Further research is needed to explore potential connections between tRNA modification systems and oxidative stress tolerance in B. thetaiotaomicron .

• What structural features of the RNA-protein complex formed by MnmG with tRNA contribute to its stability?

  Research has identified an RNA-protein complex involving MnmG that remains stable even under urea-denaturing polyacrylamide gel electrophoresis conditions . Studies involving a series of nuclease (RNase T1) and protease (trypsin) digestions, along with reverse transcription experiments, suggest that this complex may be noncovalent rather than involving covalent bonding between the protein and RNA . The precise structural determinants of this stability have not been fully elucidated, but they likely involve specific interactions between amino acid residues in MnmG and nucleotides in the tRNA substrate, particularly around the target uridine at position 34. Conserved cysteine residues in MnmG (C47 and C277 in some species) play a role in reducing FAD but appear to have additional functions in the formation of modified tRNA, as mutations of these residues prevent proper modification despite maintaining the ability to reduce FAD .

Experimental Methodology Questions

• What are the optimal conditions for expressing recombinant B. thetaiotaomicron MnmG in heterologous systems?

  Based on general principles for expressing recombinant proteins from anaerobic bacteria, optimal expression of B. thetaiotaomicron MnmG would likely require:

  Parameter	Recommended Condition	Rationale
  Expression host	E. coli BL21(DE3) or Rosetta strains	Handles proteins with rare codons
  Growth temperature	16-20°C	Slower expression improves folding
  Induction	0.1-0.5 mM IPTG	Lower concentrations reduce aggregation
  Media supplements	FAD (10-50 μM)	Ensures proper cofactor incorporation
  Oxygen conditions	Reduced oxygen tension	Mimics natural environment of B. thetaiotaomicron

  Given that B. thetaiotaomicron is a strictly anaerobic bacterium with high susceptibility to oxidative environments , expression under microaerobic or anaerobic conditions may improve proper folding and activity of recombinant MnmG. Additionally, co-expression with MnmE might be necessary for full activity testing, as these proteins function as a complex .

• How can the activity of purified recombinant MnmG be measured in vitro?

  In vitro activity of purified recombinant MnmG can be measured through several approaches:

  1. tRNA Modification Assay: Incubate purified MnmG (with MnmE) and unmodified tRNA substrates in the presence of necessary cofactors (FAD, NADH, GTP, CH2THF) and substrates (glycine, taurine, or ammonia). Detect modified tRNAs using:

     • Mass spectrometry to identify the specific modified nucleosides

     • Reverse-phase HPLC analysis of nucleosides after enzymatic digestion of tRNA

     • Gel mobility shift assays to detect tRNA structural changes

  2. Spectrophotometric Assays:

     • Monitor NAD(P)H oxidation at 340 nm

     • Track FAD reduction/oxidation by spectral changes

  3. Coupled Enzyme Assays:

     • Measure GTP hydrolysis using coupled phosphate detection systems

  4. Detection of Flavin-Iminium Intermediate:

     • Spectroscopic detection of the FADH[N5=CH2]+ intermediate formation

  The choice of glycine, taurine, or ammonia as nucleophiles will result in different modified products (cmnm^5U, τm^5U, or nm^5U, respectively), which can be distinguished by analytical methods .

• What approaches can be used to investigate the interaction between MnmE and MnmG in B. thetaiotaomicron?

  Several complementary approaches can be employed to study the MnmE-MnmG interaction:

  Approach	Methodology	Information Obtained
  Pull-down assays	Use tagged versions of MnmE or MnmG to co-purify interaction partners	Confirms direct interaction
  Surface plasmon resonance	Immobilize one protein and measure binding kinetics of the partner	Binding affinity, on/off rates
  Isothermal titration calorimetry	Measure heat changes during binding	Thermodynamic parameters
  Bacterial two-hybrid system	Use fusion constructs in reporter strain	In vivo interaction validation
  Co-immunoprecipitation	Use antibodies against one protein to precipitate complexes	Confirms interaction in cell extracts
  Size exclusion chromatography	Analyze elution profiles of individual proteins vs. mixture	Complex formation and stability
  Hydrogen-deuterium exchange MS	Compare exchange patterns of proteins alone vs. in complex	Identify interaction interfaces

  Additionally, mutations in conserved residues suspected to be at the interaction interface can be introduced to confirm their importance for complex formation and function. The interaction should be studied both in the presence and absence of substrates and cofactors (GTP, FAD, CH2THF) to understand how these factors influence complex formation .

• How do mutations in conserved MnmG cysteine residues affect enzyme function?

  Mutations in conserved MnmG cysteine residues, specifically C47 and C277 (as identified in some species), have significant effects on enzyme function. While these mutant variants can still reduce FAD, they are unable to catalyze the formation of modified tRNA . This suggests these cysteine residues play critical roles beyond just FAD reduction, possibly in:

  1. Proper positioning of the reduced FAD for reaction with methylenetetrahydrofolate

  2. Formation of the key flavin-iminium FADH[N5=CH2]+ intermediate

  3. Stabilization of the tRNA-protein complex during the modification reaction

  4. Facilitating conformational changes necessary for the complete reaction cycle

  The fact that these mutants can reduce FAD but cannot complete the modification reaction indicates these residues are involved in steps following FAD reduction, possibly in the formation or stabilization of reaction intermediates or in properly orienting the tRNA substrate for modification .

• What is the role of the flavin-iminium intermediate in tRNA modification by different MnmEG homologues?

  The flavin-iminium intermediate (FADH[N5=CH2]+) serves as a universal intermediate in tRNA modification by all MnmEG homologues, regardless of the specific nucleophile used in the reaction . This intermediate plays a crucial role in transferring the methylene group from methylenetetrahydrofolate (CH2THF) to the C5 position of the wobble uridine (U34) in target tRNAs.

  Experiments using a synthetic FADH[N5=CD2]+ analogue have demonstrated that:

  1. The FAD cofactor directly participates in the transfer of the methylene group

  2. The flavin-iminium intermediate can react with different nucleophiles:

     • With glycine to form cmnm^5U

     • With taurine to form τm^5U

     • With ammonia to form nm^5U

  This demonstrates the remarkable versatility of the MnmEG system, where a common reaction intermediate can lead to different modified nucleosides depending on the available nucleophile. The conservation of this mechanism across different bacterial species highlights its evolutionary importance in tRNA modification pathways .