Recombinant Acinetobacter sp. tRNA modification GTPase MnmE (mnmE)

What is MnmE and what is its primary function?

MnmE is a multi-domain GTPase that is highly conserved from bacteria to humans. Its primary function is to participate in the synthesis of tRNA wobble uridine modifications, specifically in the addition of a carboxymethylaminomethyl (cmnm) group at the wobble position (U34) of certain tRNAs, forming tRNA-cmnm5s2U34 . This protein works in conjunction with its partner protein MnmG in this tRNA modification pathway. The modifications are critical for proper codon recognition and translation efficiency, making MnmE an essential factor in maintaining accurate protein synthesis .

How does MnmE differ from other GTPases in terms of structure and regulation?

Unlike classical small GTP-binding proteins that are regulated by auxiliary GEFs (Guanine nucleotide Exchange Factors) and GAPs (GTPase-Activating Proteins), MnmE employs a unique regulatory mechanism. Its GTPase activity is activated through potassium-dependent homodimerization of its G domains . This mechanism of activation through dimerization represents a distinct regulatory pathway compared to the Ras superfamily of GTPases, classifying MnmE among the G proteins activated by dimerization (GADs) . MnmE also exhibits an unusually high intrinsic GTPase hydrolysis rate compared to typical Ras-like GTPases .

What is the evolutionary significance of MnmE?

MnmE is highly conserved across evolutionary boundaries from bacteria to mammals, indicating its fundamental importance in cellular processes . In eukaryotes, orthologues of MnmE and MnmG are targeted to mitochondria, suggesting their critical role in maintaining mitochondrial translation efficiency . The conservation of this protein throughout evolution underscores its essential function in tRNA modification and, consequently, in accurate protein synthesis across diverse species.

How does the MnmE-MnmG complex modify tRNA molecules?

The MnmE-MnmG complex catalyzes the addition of the carboxymethylaminomethyl (cmnm) group at the wobble position (U34) of specific tRNAs . This reaction is part of a larger pathway that modifies the uridine at position 34 to mnm5U34 in tRNAs that read codons ending in A or G . The modification process involves:

• Formation of the MnmE-MnmG complex

• GTP binding and potassium-dependent homodimerization of MnmE G domains

• GTP hydrolysis driving conformational changes

• Modification of the target uridine in the tRNA

These modifications are crucial for proper codon-anticodon interactions during translation .

Which specific tRNAs are targeted by MnmE in Acinetobacter sp.?

While the search results don't specify the exact tRNAs targeted by MnmE in Acinetobacter sp. specifically, comparative analysis with other bacterial species suggests that MnmE likely modifies tRNAs reading codons for glutamate, lysine, leucine, glutamine, and arginine . In E. coli, for instance, the MnmE/G complex targets tRNA^Glu^UUC, tRNA^Pro^UUC, and other specific tRNAs . Researchers working with Acinetobacter sp. MnmE should conduct targeted analysis to identify the specific tRNA substrates in this species through techniques such as tRNA purification followed by mass spectrometry analysis of modifications.

How do MnmE modifications affect translation efficiency?

The mnm5U34 modifications introduced by the MnmE-MnmG complex significantly impact translation in several ways:

• Codon recognition: The modifications enhance the ability of tRNAs to recognize their cognate codons efficiently

• Translation kinetics: In vitro translation studies show that MnmE modifications affect the rate of protein synthesis, particularly at specific codons

• Translocation dynamics: Modifications at the wobble position influence tRNA movement through the ribosome, affecting both A-site binding and translocation efficiency

• Reading frame maintenance: The modifications help maintain proper reading frame during translation

As demonstrated in Table 1 below (adapted from search result ), the effects of mnm5U34 modifications on translation have been experimentally verified:

What are the optimal conditions for expressing recombinant Acinetobacter sp. MnmE in heterologous systems?

Based on general principles for expressing GTPases and observations from related studies, the following protocol is recommended:

• Expression system selection: E. coli BL21(DE3) or similar strains are typically appropriate for MnmE expression . For potentially toxic proteins, consider using strains with tighter expression control.

• Vector construction: Clone the full-length mnmE gene from Acinetobacter sp. into an expression vector with an appropriate affinity tag (His6, GST, etc.) and an inducible promoter system (T7, tac).

• Expression conditions:

  • Grow cultures at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.1-0.5 mM IPTG

  • Shift temperature to 18-25°C for overnight expression to enhance proper folding

  • Supplement media with additional potassium (10-50 mM KCl) to promote stabilization of the native conformation

• Harvest and lysis:

  • Harvest cells by centrifugation at 4,000-6,000 × g

  • Resuspend in buffer containing 50 mM Tris-HCl pH 7.5-8.0, 300 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, and protease inhibitors

  • Lyse cells using sonication or pressure-based methods

Each parameter should be optimized for the specific Acinetobacter sp. MnmE variant.

What purification methods yield the highest activity for recombinant MnmE protein?

A multi-step purification protocol is recommended to obtain high-purity, active MnmE protein:

• Affinity chromatography: For His-tagged MnmE, use Ni-NTA resin with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 50 mM KCl, 5 mM MgCl2) and elution buffer (same with 250-300 mM imidazole).

• Ion exchange chromatography: Apply the protein to a Q Sepharose column equilibrated with 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, and elute with a linear gradient to 500 mM NaCl.

• Size exclusion chromatography: Use a Superdex 200 column in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 50 mM KCl, 5 mM MgCl2, and 1 mM DTT.

Throughout purification, it's essential to maintain:

• Potassium ions (critical for GTPase activity)

• Magnesium ions (required for nucleotide binding)

• Reducing agents (to prevent oxidation of cysteine residues)

• Temperature control (4°C to minimize proteolysis)

Activity verification should be performed after purification using GTPase assays to ensure functional protein recovery.

How can researchers assess the GTPase activity of purified MnmE?

Several complementary methods can be used to measure the GTPase activity of MnmE:

• Malachite green phosphate assay:

  • Mix purified MnmE (0.5-2 μM) with GTP (50-200 μM) in reaction buffer (50 mM Tris-HCl pH 7.5, 50-100 mM KCl, 5 mM MgCl2)

  • Incubate at 37°C for timed intervals

  • Stop reaction with malachite green solution

  • Measure absorbance at 630 nm to quantify released phosphate

• HPLC-based nucleotide analysis:

  • Set up reactions as above

  • Stop with EDTA or heat inactivation

  • Separate GTP and GDP using reverse-phase HPLC

  • Quantify GDP formation over time

• Continuous fluorescence-based assays:

  • Use fluorescently labeled GTP analogs

  • Monitor changes in fluorescence during GTP hydrolysis in real-time

  • Calculate kinetic parameters (kcat, Km)

• Radioactive assay:

  • Incubate MnmE with [γ-32P]GTP

  • Monitor 32Pi release by thin-layer chromatography

It's critical to include potassium controls in these assays, as MnmE's GTPase activity is potassium-dependent . Compare activity with and without potassium to demonstrate the characteristic activation mechanism.

How do conformational changes in MnmE drive the tRNA modification reaction?

The GTPase cycle of MnmE drives large-scale conformational changes that are critical for its tRNA modification function . Based on extensive structural and biochemical studies:

• GTP binding: When MnmE binds GTP in the presence of potassium ions, it undergoes dimerization of its G domains, bringing them into close proximity

• Activated state: This dimerized conformation represents the activated state of MnmE, where the catalytic machinery is correctly positioned for both GTP hydrolysis and tRNA modification

• Power stroke: GTP hydrolysis triggers a conformational "power stroke" that:

  • Repositions key catalytic residues

  • Creates appropriate binding sites for tRNA and necessary cofactors

  • Facilitates the chemical steps of the modification reaction

• Reaction coupling: The energy from GTP hydrolysis is harnessed to overcome energy barriers in the complex tRNA modification chemistry

• Cycle reset: GDP release allows MnmE to return to its initial conformation, enabling multiple catalytic cycles

This mechano-chemical coupling between GTPase activity and tRNA modification ensures the precise timing and efficiency of the reaction .

What are the implications of MnmE dysfunction for bacterial pathogenesis and antibiotic resistance?

MnmE dysfunction significantly impacts bacterial fitness and virulence through several mechanisms:

• Virulence factor expression: In pathogenic bacteria like Streptococcus pyogenes, MnmE mutants exhibit reduced expression of multiple virulence factors despite having nearly normal global transcription profiles . This reduced expression is due to impaired translation efficiencies of specific messages, including those for virulence regulators .

• Attenuated pathogenicity: MnmE pathway mutants show significant attenuation in infection models. For example, GidA/MnmE pathway mutants in S. pyogenes are highly attenuated in the murine ulcer model of soft tissue infection .

• Immune response modulation: MnmE mutants stimulate altered cytokine responses in host cells. Studies show that they induce cytokine profiles similar to wild-type bacteria but with reduced levels of tumor necrosis factor alpha and interleukin-23 .

• Potential vaccine development: The attenuated virulence combined with the preservation of antigenic targets makes MnmE pathway mutants attractive candidates for live attenuated vaccine development .

• Translational stress responses: Disruption of MnmE function creates translational stress that may interact with antibiotic stress responses, potentially affecting the development of antibiotic resistance.

These findings suggest that MnmE could be a valuable target for novel antimicrobial strategies or vaccine development efforts against Acinetobacter and other pathogenic bacteria.

How does the interplay between MnmE and MnmG coordinate tRNA modification?

The functional interaction between MnmE and MnmG represents a sophisticated molecular partnership:

• Complex formation: MnmE and MnmG form a functional heterotetrameric α2β2 complex (MnmE2-MnmG2) that serves as the active enzyme for tRNA modification

• Functional specialization:

  • MnmE contributes GTPase activity and undergoes the conformational changes that drive the reaction cycle

  • MnmG likely provides substrate binding sites and contributes specific catalytic residues for the chemical modification of tRNA

• Cofactor coordination: The complex coordinates various cofactors required for the modification chemistry, including:

  • GTP (bound by MnmE)

  • FAD (bound by MnmG)

  • 5-methyltetrahydrofolate derivatives (substrate for methyl transfer)

• Reaction orchestration: The intricate dance between these proteins involves:

  • GTP-dependent dimerization of MnmE creating an active site

  • Conformational transmission to MnmG, positioning catalytic residues

  • Coordinated binding of tRNA by both proteins

  • Synchronized chemistry at the wobble position

• Evolutionary conservation: The MnmE-MnmG partnership is conserved across species, emphasizing its fundamental importance

Understanding this interplay is crucial for developing a complete model of the tRNA modification mechanism.

What human diseases are associated with mutations in MnmE orthologues?

Mutations in human orthologues of MnmE are associated with severe mitochondrial diseases . While the search results don't provide specific details about these diseases, research indicates that:

• The human orthologue of MnmE is targeted to mitochondria, where it functions in mitochondrial tRNA modification similar to its bacterial counterpart

• Disruptions in mitochondrial tRNA modifications lead to impaired mitochondrial translation, affecting energy production and cellular metabolism

• These mutations likely contribute to a spectrum of mitochondrial disorders characterized by:

  • Neurological dysfunction

  • Myopathy

  • Metabolic abnormalities

  • Developmental delays

Researchers studying Acinetobacter sp. MnmE should consider the evolutionary conservation of this protein when exploring potential translational applications of their findings to human health.

How can structural insights from bacterial MnmE inform drug development strategies?

Structural insights into bacterial MnmE provide several avenues for antimicrobial drug development:

• Targeting the GTP binding pocket: The unique potassium-dependent GTPase mechanism of MnmE offers opportunities to design specific inhibitors that can disrupt its function without affecting human GTPases that utilize different regulatory mechanisms.

• Disrupting dimerization interfaces: Since MnmE activation requires homodimerization of its G domains , compounds that prevent this dimerization could selectively inhibit its function.

• Interfering with MnmE-MnmG complex formation: Disrupting the interaction between MnmE and MnmG would prevent formation of the functional complex required for tRNA modification .

• Exploiting conformational changes: The large-scale conformational changes that MnmE undergoes during its catalytic cycle provide potential for allosteric inhibitors that lock the protein in an inactive conformation.

• Targeting Acinetobacter-specific features: Comparative structural analysis between human and Acinetobacter MnmE may reveal species-specific structural features that could be exploited for selective targeting.

These approaches could lead to novel antibiotics against Acinetobacter and other pathogenic bacteria while minimizing effects on human cells.

What high-throughput methods can assess the impact of MnmE deficiency on the bacterial translatome?

Several sophisticated techniques can be employed to assess how MnmE deficiency affects translation on a global scale:

• Ribosome profiling:

  • This technique provides genome-wide information on ribosome positioning with nucleotide resolution

  • It can identify specific codons where ribosomes pause or stall in MnmE-deficient bacteria

  • Analysis can determine whether pausing occurs at the A site or P site, providing mechanistic insights

  • Data processing should include codon-specific analyses to identify which codons are most affected by the absence of MnmE modifications

• Proteomics with stable isotope labeling:

  • Quantitative proteomics using SILAC or similar approaches can identify proteins whose expression is altered in MnmE mutants

  • Codon bias analysis of affected proteins can reveal patterns of translational deficiency

  • This approach has successfully identified codon-specific effects in studies of other tRNA modifying enzymes

• Ribosome-bound tRNA capture:

  • This technique identifies which tRNAs are bound to ribosomes and in which site (A or P)

  • It can determine which tRNA modifications are present on ribosome-bound tRNAs

  • Comparison between wild-type and MnmE-deficient bacteria reveals how the absence of modifications affects tRNA selection by ribosomes

• Tandem-codon translation reporter assays:

  • These assays use reporter constructs with tandem repeats of specific codons

  • They provide codon-specific readouts of translation efficiency

  • They're particularly useful for identifying which codons are most affected by MnmE deficiency

These techniques provide complementary information that, when integrated, offers a comprehensive view of how MnmE-mediated tRNA modifications impact the bacterial translatome.

How can cryo-EM be used to elucidate the structural dynamics of the MnmE-MnmG-tRNA complex?

Cryo-electron microscopy (cryo-EM) offers powerful approaches to study the MnmE-MnmG-tRNA complex:

• Sample preparation strategies:

  • Prepare complexes at different stages of the GTPase/modification cycle using:

    • Non-hydrolyzable GTP analogs (GMPPNP, GTPγS) to capture pre-hydrolysis states

    • GDP·AlFx to mimic the transition state

    • GDP to capture post-hydrolysis conformations

  • Include appropriate tRNA substrates and cofactors

  • Optimize buffer conditions, including potassium concentration

• Data collection approach:

  • Collect multiple datasets representing different functional states

  • Use energy filters and phase plates to enhance contrast for smaller complexes

  • Implement motion correction algorithms to address beam-induced movement

• Classification strategies:

  • Implement 3D classification to identify distinct conformational states

  • Use focused classification to resolve dynamic regions

  • Apply time-resolved approaches if possible

• Validation and interpretation:

  • Cross-validate structures with biochemical and functional data

  • Integrate with molecular dynamics simulations to explore transitions between observed states

  • Correlate structural changes with steps in the catalytic mechanism

This approach would reveal how GTP hydrolysis by MnmE drives conformational changes that position the tRNA for modification, providing unprecedented insights into the mechanism of this complex.

What are the most effective approaches for conducting structure-guided mutagenesis of MnmE to dissect its mechanism?

Structure-guided mutagenesis of MnmE should follow a systematic approach to dissect distinct aspects of its mechanism:

• Target selection strategy:

  • GTP binding site residues: Mutate residues in the G1-G5 loops that contact GTP

  • Dimerization interface residues: Target amino acids at the G domain interface

  • Potassium coordination site: Modify residues that coordinate the potassium ion

  • Switch regions: Alter residues in switch I and II that undergo conformational changes

  • MnmG interaction surface: Mutate residues at the MnmE-MnmG interface

  • tRNA binding residues: Identify and modify potential tRNA contact points

• Mutation design principles:

  • Use conservative substitutions to probe specific interactions

  • Create charge reversals to disrupt electrostatic interactions

  • Engineer disulfide bridges to restrict conformational flexibility

  • Introduce bulky side chains to block binding interfaces

• Functional characterization workflow:

  • GTPase activity assays: Measure intrinsic and potassium-stimulated GTP hydrolysis

  • Dimerization assays: Use size exclusion chromatography, FRET, or analytical ultracentrifugation

  • tRNA binding studies: Employ electrophoretic mobility shift assays or surface plasmon resonance

  • tRNA modification assays: Quantify modification efficiency using mass spectrometry

  • In vivo complementation: Test ability to rescue MnmE knockout phenotypes

• Integrated analysis framework:

  • Correlate structural features with biochemical phenotypes

  • Identify residues critical for specific steps in the mechanism

  • Build a comprehensive model of the structure-function relationship

This approach would provide mechanistic insights at amino acid resolution into how MnmE functions in tRNA modification.