Recombinant Acinetobacter sp. tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG (mnmG), partial

What is the fundamental role of MnmG in bacterial tRNA modification systems?

MnmG (also called GidA) functions as part of an evolutionarily conserved bacterial protein complex with MnmE to install a 5-carboxymethylaminomethyl (cmnm⁵) or a 5-taurinomethyl (τm⁵) group onto wobble uridines (position 34) of several tRNA species. This modification is crucial for proper and efficient protein translation, particularly for maintaining translational fidelity in mixed codon family boxes. The MnmEG complex represents the first step in the biosynthesis of xm⁵U modifications in bacteria, which may be further processed by additional enzymes like MnmC to generate other modified nucleosides such as 5-methylaminomethyl (mnm⁵) .

What cofactors are essential for MnmG activity and how do they contribute to its function?

MnmG specifically binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH), while its partner MnmE binds guanosine-5′-triphosphate (GTP) and methylenetetrahydrofolate (CH₂THF). These cofactors are critical for the catalytic activity of the complex. The FAD bound to MnmG plays a particularly crucial role as it forms a flavin-iminium intermediate (FADH[N⁵=CH₂]⁺) that serves as a methylene carrier in the reaction mechanism. Recent research has demonstrated that this flavin-iminium intermediate functions as a universal intermediate for all MnmEG homologues, transferring the methylene group from CH₂THF to the C5 position of U₃₄ in the target tRNAs .

How do MnmG and MnmE interact to form a functional complex?

Small-angle X-ray scattering (SAXS) analysis has revealed that MnmE and MnmG form an α₂β₂ complex in an asymmetric manner, which differs from earlier proposed models. This complex consists of one MnmE dimer bound to one MnmG dimer. Interestingly, this interaction appears to be dynamic and dependent on the nucleotide-binding state. When GTP is bound, size exclusion chromatography (SEC) experiments coupled with multiangle light scattering (SEC-MALS) and SAXS indicate that MnmE and MnmG form a higher oligomeric state (α₄β₂, representing two MnmE dimers binding to one MnmG dimer). This oligomerization appears to be reversible upon GTP hydrolysis, suggesting that nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle .

What evidence supports the role of flavin-iminium FADH[N⁵=CH₂]⁺ as a central intermediate in the MnmEG reaction?

Recent biochemical studies have provided multiple lines of evidence supporting flavin-iminium FADH[N⁵=CH₂]⁺ as a key intermediate in the MnmEG reaction:

• Experiments using a synthetic FADH[N⁵=CD₂]⁺ analogue (containing deuterium atoms) demonstrated the intermediacy of FAD in transferring the methylene group from CH₂THF to the C5 position of U₃₄.

• When MnmEG reactions containing the deuterated flavin-iminium intermediate were conducted with alternate nucleophiles such as taurine and ammonia, they led to the formation of the anticipated U₃₄-modified tRNAs.

• HPLC-HRMS analysis showed that when CH₂THF was the methylene source, the fully modified cmnm⁵s²U nucleoside was observed with an m/z of 348.08. When FADH[N⁵=CD₂]⁺ was used as the methylene source, the peak shifted to 350.09, corresponding to cmnm⁵s²U-d₂, confirming the transfer of the deuterated methylene group.

These findings unambiguously demonstrate that FAD[N⁵=CH₂]⁺ serves as the universal intermediate for all MnmEG homologues, regardless of the final nucleophile used in the reaction .

What is known about the kinetic properties of the MnmEG-catalyzed reactions?

The MnmEG complex exhibits distinct kinetic properties compared to other enzymes in the tRNA modification pathway. The following table compares the kinetic parameters of MnmEG with those of the MnmC enzyme and its domains:

What techniques are most effective for investigating the MnmEG complex structure and interactions?

Several complementary techniques have proven effective for studying the MnmEG complex:

• Small-angle X-ray scattering (SAXS) has been successfully used to unravel the mode of interaction between MnmE and MnmG in the α₂β₂ complex, revealing an asymmetric interaction distinct from previously proposed models.

• Size exclusion chromatography (SEC) combined with multiangle light scattering (SEC-MALS) provides valuable information about the oligomeric state of the MnmEG complex, particularly the GTP-dependent formation of higher-order structures.

• Urea-denaturing polyacrylamide gel electrophoresis can be used to identify stable RNA-protein complexes, which may be relevant to the catalytic mechanism.

• Nuclease (RNase T1) and protease (trypsin) digestions combined with reverse transcription experiments help characterize the nature of RNA-protein interactions, distinguishing between covalent and non-covalent associations.

• HPLC-HRMS (High-Performance Liquid Chromatography-High Resolution Mass Spectrometry) is invaluable for analyzing modified nucleosides and monitoring reaction products, allowing detection of mass shifts that provide mechanistic insights .

How can researchers assess the tRNA substrate specificity of MnmG?

Studies have shown that MnmG exhibits substrate specificity for certain tRNA species. To assess this specificity, researchers can:

• Use chimeric tRNA constructs: Create chimeric versions of tRNAs by inserting the gene of interest into a scaffold tRNA (such as human cytosolic tRNA lacking the anticodon region), resulting in RNA containing the complete bacterial sequence fused at its 5′- and 3′-ends to the truncated anticodon stem.

• Analyze HPLC profiles of modified tRNAs: HPLC analysis of tRNA hydrolysates can differentiate between various modified nucleosides (cmnm⁵s²U, nm⁵s²U, mnm⁵s²U) and their non-thiolated counterparts.

• Use tRNA from specific mutant strains: For example, analyzing tRNA from a ΔtrmL strain (in which methylation of the ribose in the wobble uridine is impaired) can help identify specific modifications.

Research has shown distinct modification patterns for different tRNA species. For instance, tRNAᴳˡʸ contains cmnm⁵U but shows no traces of mnm⁵U or nm⁵U, suggesting it is not modified by the ammonium-dependent MnmEG pathway and is not a substrate for MnmC(o) .

What in vitro reconstitution systems can be used to study MnmG activity?

In vitro reconstitution systems for studying MnmG activity typically include:

• Purified recombinant MnmE and MnmG proteins: These can be expressed separately and combined to form the active complex.

• Essential cofactors: Including FAD, NADH (for MnmG), GTP, and CH₂THF (for MnmE).

• tRNA substrate: Either in vitro transcribed tRNAs or tRNAs purified from appropriate knockout strains (e.g., mnmC null mutant).

• Appropriate buffer conditions: Often containing Mg²⁺ (5 mM), which is required for GTP hydrolysis by MnmE.

• Alternative substrates: Depending on the specific modification pathway being studied, researchers may include glycine (for cmnm⁵ formation), ammonia (for nm⁵ formation), or taurine (for τm⁵ formation).

For optimal MnmEG activity assays, the reaction conditions should be distinct from those used for MnmC reactions, as MnmC activities are known to be severely inhibited at 5 mM Mg²⁺ .

How does the genomic context of mnmG differ in Acinetobacter compared to other bacterial species?

In Acinetobacter species, the genomic context of mnmG (also called gidA) differs from that observed in Escherichia coli. In E. coli, the origin of replication (oriC) maps between the gidA (mnmG) and mioC genes. In contrast, Acinetobacter spp. for which genome sequences have been completed contain a single circular chromosome with the origin of replication located between the rpmH and dnaA loci, an arrangement also seen in Pseudomonas spp. This places mnmG in a different context relative to the replication origin compared to E. coli .

This difference in genomic arrangement may have implications for the coordinated expression of mnmG with other genes involved in DNA replication or protein synthesis, potentially reflecting distinct regulatory mechanisms in Acinetobacter species. The conservation of this arrangement across sequenced Acinetobacter genomes suggests it has functional significance in these organisms .

What potential role might MnmG play in Acinetobacter pathogenicity or antibiotic resistance?

While direct evidence linking MnmG to Acinetobacter pathogenicity is limited in the provided search results, there are several reasons why MnmG might be relevant to pathogenicity and antibiotic resistance in Acinetobacter:

• Acinetobacter species, particularly A. baumannii, have become important sources of hospital-acquired infections due to their remarkable ability to acquire antibiotic resistance determinants.

• The essential processes of chromosomal DNA replication, transcription, and cell division are attractive targets for the rational design of antimicrobial drugs against Acinetobacter spp.

• As MnmG is involved in tRNA modification, which is crucial for translational fidelity, disruptions in MnmG function could potentially affect the expression of virulence factors or antibiotic resistance determinants.

• Acinetobacter species show several key differences from other pathogenic gammaproteobacteria, particularly in global stress response pathways, which may involve translation-related mechanisms.

The connection between tRNA modifications and stress responses could potentially link MnmG function to survival mechanisms that contribute to Acinetobacter's notable ability to persist in hospital environments and acquire resistance .

What unresolved questions remain about the catalytic mechanism of MnmG?

Despite significant advances in understanding MnmG function, several questions remain unresolved:

• Nature of the RNA-protein complex: While studies have identified an RNA-protein complex stable to urea-denaturing polyacrylamide gel electrophoresis, the exact nature of this complex (whether covalent or non-covalent) and its role in the catalytic mechanism remain to be fully elucidated.

• Precise role of conserved cysteines: Although conserved MnmG cysteine residues C47 and C277 have been shown to reduce FAD, their inability to promote modified tRNA formation suggests more complex roles that need further investigation.

• Mechanism of methylene transfer: Two competing models exist for how the methylene moiety might be transferred to the tRNA base - one involving a cysteine-mediated nucleophilic attack and another where N1 assists the transfer without forming an enzyme-RNA covalent complex.

• Conformational changes during catalysis: The relationship between GTP-dependent oligomerization states and the catalytic cycle requires further clarification.

• Species-specific variations: The extent to which the mechanism of MnmG varies across bacterial species, particularly in pathogens like Acinetobacter, remains to be fully explored .

How might MnmG be targeted for the development of new antimicrobial agents against Acinetobacter species?

As antibiotic resistance in Acinetobacter species continues to pose a serious clinical challenge, MnmG represents a potential target for novel antimicrobial development for several reasons:

• MnmG is involved in the essential process of tRNA modification, which is crucial for proper protein translation.

• The essential processes of chromosomal DNA replication, transcription, and cell division (which rely on accurate translation) are attractive targets for the rational design of antimicrobial drugs.

• The unique features of the MnmEG complex, including its dependence on multiple cofactors (FAD, NADH, GTP, CH₂THF) and complex catalytic mechanism, offer multiple potential points of intervention.

• The differences in genomic context and potentially in regulatory mechanisms between Acinetobacter and other bacterial species might allow for the development of more selective inhibitors.

• The flavin-iminium intermediate represents a unique aspect of the MnmEG mechanism that could be specifically targeted.

Future research efforts could focus on high-throughput screening for small molecule inhibitors of MnmG activity, structure-based drug design targeting the active site or protein-protein interaction surfaces, or development of molecules that interfere with the formation of the flavin-iminium intermediate .