Recombinant Coxiella burnetii (Dimethylallyl)adenosine tRNA methylthiotransferase MiaB (miaB)

What is the structural composition of Coxiella burnetii MiaB?

Coxiella burnetii MiaB, like other characterized MiaB proteins, is likely composed of three distinct domains with specific functions. Based on structural analysis of MiaB proteins, these domains include an N-terminal MTTase domain containing cysteine residues (Cys27, Cys63, Cys97) that coordinate three of the irons in the auxiliary [Fe4S4] cluster, an RS domain with a conserved core fold consisting of a shortened (βα)6 triosephosphate isomerase (TIM) barrel that contains additional cysteine residues (Cys171, Cys173, Cys178) coordinating another [Fe4S4] cluster, and a C-terminal TRAM domain involved in tRNA recognition and binding . The protein's functional activity depends on proper coordination of these iron-sulfur clusters, which are critical for the radical SAM-dependent catalytic mechanism. These structural elements work together to perform the methylthiolation of specific tRNA nucleosides.

What is the primary function of tRNA methylthiotransferase MiaB in bacterial systems?

MiaB functions as a radical SAM enzyme responsible for the post-transcriptional modification of transfer RNAs (tRNAs), specifically catalyzing the methylthiolation of N6-isopentenyladenosine (i6A37) to form 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 of certain tRNAs . This modification plays vital roles in translation accuracy and efficiency. The MiaB reaction takes place in two distinct half-reactions utilizing S-adenosylmethionine (SAM) as both a radical generator and methyl donor . These modifications are crucial for proper codon-anticodon interactions and contribute to translational fidelity. The conserved nature of this enzyme across bacterial species, including pathogens like C. burnetii, suggests its fundamental importance in bacterial physiology and potentially in pathogenesis.

How are recombinant C. burnetii proteins typically expressed and purified for research purposes?

Recombinant C. burnetii proteins are typically overexpressed in E. coli expression systems using histidine-tagged fusion constructs. As demonstrated in immunization experiments, genes encoding target C. burnetii proteins (such as Omp, Pmm, HspB, Fbp, Orf410, Crc, CbMip, and MucZ) are cloned into appropriate expression vectors, transformed into E. coli, and expression is induced under controlled conditions . The recombinant proteins are then purified using affinity chromatography, typically utilizing the His-tag for nickel affinity purification. For instance, in studies examining recombinant C. burnetii proteins for vaccine development, the proteins were "overexpressed in E. coli as his-tagged fusion proteins and partially purified" . Additional purification steps may include ion exchange chromatography or gel filtration to achieve higher purity. Quality control typically involves SDS-PAGE and Western blotting to confirm protein identity and purity before proceeding to functional assays or immunological studies.

What techniques are used to assess the immunogenicity of recombinant C. burnetii proteins?

Multiple techniques are employed to evaluate the immunogenicity of recombinant C. burnetii proteins. In mouse model studies, proteins are typically administered as mixtures with appropriate adjuvants, followed by collecting serum samples at regular intervals to assess antibody production . ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to detect and quantify specific antibody responses. For example, in recombinant protein ELISA protocols, plates are coated with the recombinant antigen (typically at 1 ng/μL concentration in carbonate-bicarbonate buffer), blocked with casein or similar blocking agents, and then incubated with diluted serum samples . Detection utilizes species-specific secondary antibodies conjugated to enzymes like horseradish peroxidase, followed by colorimetric development with substrates such as TMB (3,3',5,5'-tetramethylbenzidine) . Western blotting provides complementary analysis to confirm antibody specificity, where proteins are separated by SDS-PAGE, transferred to membranes, and probed with serum followed by detection using conjugated secondary antibodies and visualization reagents like diaminobenzidine .

What role does substrate recognition play in MiaB function?

Substrate recognition is critical for MiaB function and involves specific interactions between the enzyme domains and the target tRNA. Based on structural studies, MiaB shows "exquisite selectivity for i6A37," with all three domains contributing to RNA binding . The TRAM domain contains conserved amino acids that specifically recognize U33, which is conserved in all tRNAs. This recognition involves a positively charged pocket formed by residues such as Lys409, Arg410, and Lys451, which provide selectivity for the U33 base . Additionally, the RS domain contributes two phenylalanine residues that recognize A36, which is found in all tRNAs that undergo the ms2i6A modification. These residues selectively bind A36 while excluding other nucleotides at this position. The enzyme specifically recognizes i6A37 predominantly through its isopentenyl modification, which explains why MiaB cannot modify unmodified A37 . This precise substrate recognition mechanism ensures that the methylthiolation reaction occurs only on the appropriate tRNA substrates at the correct position.

What challenges exist in expressing and purifying active recombinant C. burnetii MiaB?

Expressing and purifying active recombinant C. burnetii MiaB presents multiple technical challenges that researchers must address. The primary obstacle involves maintaining the integrity of the iron-sulfur clusters essential for enzymatic activity. MiaB contains two [Fe4S4] clusters coordinated by specific cysteine residues in the MTTase and RS domains . These clusters are extremely oxygen-sensitive, requiring anaerobic conditions during expression, purification, and storage to prevent oxidative damage and loss of activity. Additionally, the incorporation of iron-sulfur clusters often requires co-expression with iron-sulfur assembly machinery proteins or supplementation with iron and sulfur sources during expression. Another significant challenge is protein solubility, as recombinant proteins from C. burnetii frequently form inclusion bodies in E. coli expression systems. This necessitates optimization of expression conditions (temperature, induction time, strain selection) or the development of refolding protocols. Finally, assessing the activity of purified MiaB requires specialized assays using in vitro transcribed tRNA substrates and analytical techniques such as HPLC-MS to detect methylthiolated products, adding complexity to functional verification.

How can we assess the enzymatic activity of recombinant MiaB in vitro?

Assessing the enzymatic activity of recombinant MiaB requires sophisticated biochemical approaches that recapitulate the two-step radical SAM mechanism. A comprehensive in vitro activity assay would include: (1) Substrate preparation - obtaining properly folded tRNA substrates containing i6A37, either through in vitro transcription followed by enzymatic installation of the isopentenyl group or isolation from appropriate genetic backgrounds; (2) Reaction conditions - establishing an anaerobic environment with reducing agents (like dithionite) to maintain the iron-sulfur clusters in their active state, along with SAM as the methyl donor and radical initiator, and frequently sodium dithionite as an electron source; (3) Analytical detection - utilizing mass spectrometry to detect and quantify the formation of ms2i6A37 in the tRNA substrate . Liquid chromatography-mass spectrometry (LC-MS) approaches are particularly valuable, as demonstrated in studies of other proteins where "data were acquired by spraying the sample at 3.4 kV capillary voltage in the Q-TOF detector and reading spectra from alternating scans at low and high collision energies" . Time-course experiments can provide insights into reaction kinetics, while site-directed mutagenesis of conserved residues can identify those critical for catalysis.

What structural elements determine substrate specificity in MiaB and how might this differ in C. burnetii?

The substrate specificity of MiaB enzymes is determined by several key structural elements that could have specific adaptations in the C. burnetii ortholog. Based on structural studies of MiaB proteins, the TRAM domain plays a crucial role through a positively charged pocket lined by conserved residues (Lys409, Arg410, Lys451) that specifically recognize the U33 base conserved in target tRNAs . This "unique binding mode" involves immersion of the U33 base in this pocket, with additional coordination through water molecules mediated by Ser408 and Gln414. The hydrophobic interactions between C5 and C6 of U33 with Val426 and Thr449 further contribute to specificity . Additionally, the RS domain contributes two phenylalanine residues that specifically recognize A36, preventing the modification of tRNAs with other nucleotides at this position. The recognition of i6A37 occurs "predominantly by its isopentenyl modification," explaining why MiaB cannot modify unmodified A37 . In C. burnetii, sequence variations in these recognition regions might confer slightly different substrate preferences or recognition patterns that could be adapted to the specific tRNA pool of this intracellular pathogen.

How does temperature and pH affect the stability and activity of recombinant C. burnetii MiaB?

The stability and activity of recombinant C. burnetii MiaB likely have distinct temperature and pH profiles reflecting the unique lifestyle of this pathogen. C. burnetii replicates within acidified lysosome-like compartments with pH values around 4.5-5.0, suggesting its proteins may be adapted to function in acidic environments. While specific data for C. burnetii MiaB is not provided in the search results, we can hypothesize that the protein may exhibit optimal activity at slightly acidic pH compared to orthologs from neutralophilic bacteria. Temperature sensitivity is another critical factor, as C. burnetii experiences various temperatures during its lifecycle. The thermostability of its MiaB would be important for maintaining consistent tRNA modification during temperature fluctuations. The iron-sulfur clusters central to MiaB function are particularly susceptible to environmental conditions, with both extreme pH and temperature potentially affecting cluster stability and enzyme activity. Experimental approaches to determine these parameters would include thermal shift assays to measure protein unfolding at different temperatures, circular dichroism spectroscopy to monitor secondary structure changes, and enzymatic activity assays conducted across pH and temperature ranges. These stability profiles would be especially important when designing expression and purification protocols for the recombinant protein and when establishing in vitro activity assays.

What expression systems are optimal for producing active recombinant MiaB proteins?

For producing active recombinant MiaB proteins, specialized expression systems that facilitate iron-sulfur cluster assembly are essential. E. coli strains engineered to enhance iron-sulfur protein expression, such as SufFeScient cells or those co-expressing the iron-sulfur cluster (ISC) assembly machinery, provide significant advantages over standard laboratory strains. Expression vectors should include tightly regulated promoters (like T7-lac) to control expression rates, as slower expression often improves proper folding and cofactor incorporation. Based on previous recombinant C. burnetii protein work, where genes were "overexpressed in E. coli as his-tagged fusion proteins" , fusion tags must be carefully selected, with His-tags being common for purification but potentially affecting iron-sulfur cluster coordination. Expression conditions require significant optimization, with lower temperatures (16-20°C) and longer induction periods favoring proper folding. Critically, anaerobic expression systems or addition of iron sources (ferrous ammonium sulfate) and sulfur sources (cysteine) to the growth medium can enhance iron-sulfur cluster incorporation. For purification, all buffers must contain reducing agents (DTT, β-mercaptoethanol, or dithionite) and should ideally be conducted in anaerobic chambers to prevent cluster degradation. Success can be monitored by the characteristic brown coloration of properly assembled iron-sulfur proteins and UV-visible spectroscopy to confirm cluster presence.

How can site-directed mutagenesis be used to study MiaB function in C. burnetii?

Site-directed mutagenesis represents a powerful approach to dissect the structure-function relationships of C. burnetii MiaB. Based on structural data from MiaB proteins showing conserved domains and critical residues, researchers should target several categories of amino acids: (1) Iron-sulfur cluster coordination sites - mutating the conserved cysteine residues (likely corresponding to positions similar to Cys27, Cys63, Cys97 in the MTTase domain and Cys171, Cys173, Cys178 in the RS domain ) to serine or alanine would disrupt cluster binding while minimizing structural perturbation; (2) tRNA recognition residues - mutating amino acids in the TRAM domain that interact with the conserved U33 nucleotide (corresponding to positions like Lys409, Arg410, Lys451 ) would alter substrate recognition; (3) Catalytic residues - targeting amino acids involved in SAM binding and radical generation. The mutant constructs should be generated using PCR-based methods and confirmed by sequencing before expression and purification as described above. Functional characterization would involve comparative analysis of wild-type and mutant proteins, assessing changes in tRNA binding affinity using techniques like electrophoretic mobility shift assays, methylthiolation activity using mass spectrometry-based assays, and structural integrity using circular dichroism or thermal shift assays. Complementation studies in MiaB-deficient bacterial systems could provide additional insights into the functional significance of specific residues.

What methods can be used to compare MiaB from C. burnetii with orthologs from other bacterial species?

Comprehensive comparison of MiaB from C. burnetii with orthologs from other bacterial species requires a multi-faceted approach combining bioinformatic analysis with biochemical and structural characterization. Sequence alignment tools should be applied to compare C. burnetii MiaB with characterized orthologs (including those from Psychrobacter sp. and Streptomyces coelicolor mentioned in the search results ) to identify conserved domains, catalytic residues, and potential species-specific variations. Homology modeling based on existing MiaB structures can predict structural differences that might influence substrate recognition or catalytic efficiency. For experimental comparisons, recombinant proteins from multiple species should be expressed and purified under identical conditions to control for methodological variations. Enzymatic assays comparing substrate specificity, reaction kinetics, and cofactor requirements would reveal functional differences. Thermal stability assays and pH-activity profiles could identify adaptations to different environmental niches, particularly relevant for C. burnetii which survives in acidified compartments. Structural studies using techniques like X-ray crystallography or cryo-electron microscopy would provide direct visualization of structural differences. Cross-species complementation experiments, where the C. burnetii miaB gene is expressed in other bacterial species lacking their native miaB (or vice versa), would test functional conservation and identify species-specific roles.

What are the key considerations for developing in vitro assays to measure MiaB activity?

Developing robust in vitro assays for MiaB activity requires careful consideration of multiple factors to recapitulate the complex radical SAM-based catalytic mechanism. The substrate preparation is critical - researchers must generate appropriate tRNA substrates already containing the i6A37 modification, as MiaB specifically recognizes "i6A37 predominantly by its isopentenyl modification" . This typically requires either enzymatic treatment of in vitro transcribed tRNAs or isolation from appropriate genetic backgrounds. Reaction conditions must be strictly anaerobic, as the iron-sulfur clusters are oxygen-sensitive, requiring the use of anaerobic chambers or Schlenk line techniques. Essential components include S-adenosylmethionine (SAM) serving as both radical generator and methyl donor, a suitable electron source (typically sodium dithionite), and buffer conditions mimicking physiological pH and salt concentrations. For the C. burnetii enzyme specifically, considering the acidic environment of its natural niche may be relevant. Detection methods must be sensitive enough to monitor the methylthiolation reaction, with mass spectrometry being the gold standard for directly identifying the ms2i6A modification. HPLC separation coupled to mass spectrometry allows quantification of reaction progress over time. Controls must include heat-inactivated enzyme, reactions lacking electron donors, and assays with mutant tRNAs lacking the target nucleotide to confirm specificity.

How could recombinant C. burnetii MiaB be utilized in vaccine development research?

While direct evidence linking C. burnetii MiaB to vaccine development is not present in the search results, several strategic approaches could be considered based on experiences with other recombinant C. burnetii proteins. Previous immunization experiments with recombinant C. burnetii proteins showed that most tested proteins "proved to be antigenic in BALB/c mice when administered as protein mixtures" , suggesting MiaB could potentially elicit an immune response if properly formulated. A comprehensive vaccine development approach would first require evaluating MiaB immunogenicity through administration to animal models with appropriate adjuvants, followed by measuring antibody production using ELISA methods similar to those described for other C. burnetii proteins, where plates are "coated with the recombinant protein diluted at a concentration of 1 ng/μL" . If immunogenic, protective efficacy would be assessed through challenge studies with virulent C. burnetii, evaluating parameters such as bacterial burden, organ pathology (particularly "spleen and liver weights" ), and survival rates. Additionally, a differential approach could be developed that distinguishes vaccinated from infected animals, similar to the observation that "r-Ybgf antigen induced by vaccination" showed different serological patterns compared to natural infection, which "may be very useful given the absence of a protocol for the differentiation of infected from vaccinated animals" . The potential advantage of targeting MiaB would be its conserved nature and essential function, though this must be balanced against accessibility to antibodies during infection.

What are the main unanswered questions regarding C. burnetii MiaB structure and function?

Several critical knowledge gaps exist regarding C. burnetii MiaB structure and function that present opportunities for future research. First, the precise atomic structure of C. burnetii MiaB remains undetermined, unlike structural data available for MiaB from other organisms where "structures of an MTTase in the presence of its macromolecular substrate" have been resolved . Second, the specific tRNA substrates preferentially modified by C. burnetii MiaB are unknown - while MiaB generally modifies i6A37-containing tRNAs, species-specific preferences may exist that could relate to the unique codon usage patterns of this pathogen. Third, the regulation of MiaB expression and activity during different stages of C. burnetii's intracellular lifecycle remains unexplored, particularly how its activity might respond to the acidified environment of the Coxiella-containing vacuole. Fourth, potential moonlighting functions beyond canonical tRNA modification haven't been investigated - some bacterial enzymes perform secondary functions unrelated to their primary catalytic activity. Fifth, the impact of MiaB-catalyzed tRNA modifications on C. burnetii virulence gene expression requires systematic study, particularly whether specific virulence factors depend on optimally modified tRNAs for efficient translation. Finally, the immunological significance of MiaB during infection is unknown - whether it generates specific antibody responses in infected hosts and whether these antibodies might neutralize bacterial functions.

How might high-throughput screening approaches be used to identify inhibitors of C. burnetii MiaB?

High-throughput screening for C. burnetii MiaB inhibitors would require innovative assay development to accommodate the complex radical SAM chemistry. A primary screening platform could utilize a simplified activity assay measuring SAM cleavage rather than complete tRNA modification, as the radical generation step is essential for function. This could be monitored through formation of 5'-deoxyadenosine (5'-dA) using fluorescence-based detection methods. Alternative approaches might include thermal shift assays to identify compounds that bind MiaB and alter its thermal stability profile. Once established, diverse compound libraries could be screened, including focused collections targeting iron-sulfur proteins or nucleoside-binding enzymes. Natural product libraries might be particularly valuable, as many antibiotics target bacterial RNA metabolism. Hit compounds would undergo validation through concentration-response testing and counterscreens against human radical SAM enzymes to establish selectivity. Secondary assays would confirm activity against the complete tRNA methylthiolation reaction using mass spectrometry detection. Medicinal chemistry optimization could then enhance potency, selectivity, and pharmacokinetic properties. Finally, cellular testing would confirm inhibitor activity in C. burnetii-infected cell models, monitoring bacterial growth and survival. The development of such inhibitors could provide valuable chemical probes to study MiaB function in C. burnetii and potentially lead to novel therapeutic approaches for Q fever infections.

How do the iron-sulfur clusters in MiaB impact experimental design for functional studies?

The presence of oxygen-sensitive iron-sulfur clusters in MiaB creates significant experimental design challenges for functional studies. Researchers must implement strict anaerobic techniques throughout all experimental procedures, from protein expression to activity assays. For expression, consider utilizing specialized E. coli strains with enhanced iron-sulfur assembly machinery and conducting growth under low-oxygen conditions with iron and sulfur supplementation. Purification requires anaerobic chambers or Schlenk line techniques, with all buffers containing reducing agents and being thoroughly degassed. Spectroscopic characterization is essential to confirm cluster integrity, typically using UV-visible spectroscopy to observe the characteristic absorbance of [Fe4S4] clusters around 400 nm, while electron paramagnetic resonance can detect cluster oxidation states. For activity assays, all components must be made anaerobic, and reactions conducted in sealed vessels under inert gas. When designing mutagenesis studies, researchers should carefully distinguish between mutations affecting cluster coordination versus those impacting substrate binding or catalysis, as cluster disruption will abolish all activity regardless of the specific mechanism. During data interpretation, researchers must consider whether observed effects result from direct inhibition of catalysis versus indirect effects on cluster stability. Finally, when translating in vitro findings to cellular contexts, consider how the oxidizing environment inside eukaryotic host cells might affect MiaB activity during C. burnetii infection.

What comparative genomic approaches could reveal evolutionary insights about C. burnetii MiaB?

Comparative genomic approaches offer powerful tools to understand the evolutionary history and functional adaptations of C. burnetii MiaB. A comprehensive analysis would begin with identifying MiaB orthologs across diverse bacterial phyla using both sequence similarity searches and conserved domain architecture recognition. Phylogenetic tree construction would place C. burnetii MiaB in evolutionary context, potentially revealing horizontal gene transfer events or unusual evolutionary patterns. Synteny analysis examining the genomic neighborhoods of miaB genes across species could identify functionally related genes that might form operons or regulatory networks. Selective pressure analysis using dN/dS ratios would identify regions under positive or purifying selection, highlighting functionally critical residues versus those potentially adapting to specific environments. Comparison of C. burnetii MiaB with orthologs from related intracellular pathogens versus free-living bacteria could reveal adaptations specific to intracellular lifestyle. Correlative analysis with tRNA gene content and codon usage patterns across species might uncover co-evolution between MiaB and its tRNA substrates. Finally, comparative structural modeling based on known MiaB structures would predict C. burnetii-specific structural adaptations, particularly in substrate binding regions. Together, these approaches could reveal how C. burnetii MiaB has potentially evolved specialized features adapted to the unique environmental niche of this obligate intracellular pathogen.