Recombinant Bordetella bronchiseptica Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the structural composition of full-length Bordetella bronchiseptica MtgA?

Full-length Bordetella bronchiseptica MtgA is a 242 amino acid protein with an N-terminal transmembrane (TM) domain and a C-terminal catalytic domain. The amino acid sequence (MPKPTARRLNWFRVITAVIMAVLCIAILYQLWMFSLVVWYAYRDPGSSAIMRQELARLRE RDPEAELKYQWVPYDRISNTLKQAVVASEDANFTEHDGVEWDAIRKAWEYNQRQAERGRT KMRGGSTITQQLAKNLFLSGSRSYLRKGQELVLAYMIEHVMPKERILELYLNVAEWGVGV FGAEAAARHYYNTSAARLGAGQAARLAAMLPNPRYYDRHRNTGYLNSRTATLTRRMRMVE IP) includes hydrophobic residues in the N-terminal region that anchor the protein to the membrane . Studies with other bacterial MtgA proteins have demonstrated that the TM segment significantly influences enzymatic activity, with full-length proteins showing higher activity than truncated forms lacking the TM domain .

How does the transmembrane domain affect MtgA's enzymatic function?

The transmembrane segment of MtgA plays a critical role in its enzymatic function. Research has demonstrated that full-length MtgA proteins containing their native transmembrane domains exhibit significantly higher glycosyltransferase activity compared to truncated forms without these domains . This phenomenon has been observed across multiple bacterial species, including Streptococcus pneumoniae PBP2a, where the TM domain influences the length of glycan chains produced .

The mechanism behind this enhanced activity likely involves:

• Proper orientation of the enzyme at the cytoplasmic membrane where peptidoglycan synthesis occurs

• Facilitation of substrate binding, particularly lipid II, which contains a hydrophobic moiety

• Potential interactions with other membrane-associated cell wall synthesis machinery

• Influence on the binding of inhibitors such as moenomycin

For experimental purposes, this indicates that using full-length recombinant protein may yield more physiologically relevant results compared to studies using only the catalytic domain.

What expression systems are optimal for producing recombinant B. bronchiseptica MtgA?

Successful expression of recombinant B. bronchiseptica MtgA can be achieved in E. coli expression systems with appropriate modifications to accommodate the transmembrane domain . The following methodological approach is recommended:

• Vector selection: Vectors containing an N-terminal His-tag facilitate downstream purification while preserving the natural C-terminus. The commercially available recombinant protein utilizes this configuration .

• Expression conditions:

  • Induction at lower temperatures (16-20°C) often improves folding of membrane-associated proteins

  • IPTG concentration should be optimized (typically 0.1-0.5 mM)

  • Extended expression times (16-20 hours) at reduced temperatures may improve yield

• Cell lysis considerations:

  • Detergent selection is critical for solubilizing the transmembrane domain

  • Mild non-ionic detergents (DDM, CHAPS) at concentrations just above CMC are preferred

  • Inclusion of glycerol (10-15%) helps stabilize the protein

• Alternative approaches:

  • For difficult expressions, fusion partners like MBP or SUMO may improve solubility

  • Cell-free expression systems can be considered for transmembrane proteins

This approach has been validated for the commercial recombinant product, resulting in >90% purity as determined by SDS-PAGE analysis .

What methods are available for measuring MtgA glycosyltransferase activity?

Several complementary approaches can be employed to assess MtgA glycosyltransferase activity:

• Fluorescent substrate assays:

  • Dansylated or NBD-labeled lipid II substrates can be used to monitor glycan polymerization

  • Fluorescently labeled moenomycin derivatives enable high-throughput screening of inhibitors and substrate binding

  • Detection of FRET (Förster resonance energy transfer) between appropriately labeled substrates provides real-time monitoring

• Radiolabeled substrate incorporation:

  • [³H]-labeled substrates allow quantitative measurement of glycan chain formation

  • Similar to methods used in B. pertussis peptidoglycan studies where [³H]diaminopimelic acid (DAP) was utilized to specifically label peptidoglycan

• LC-MS based approaches:

  • Analysis of reaction products by HPLC or LC-MS enables detailed characterization of glycan products

  • Can determine glycan chain length and modifications

• Functional complementation:

  • Expression of B. bronchiseptica MtgA in bacterial strains with conditional mutations in endogenous glycosyltransferases

When designing these assays, it's important to consider that full-length MtgA with intact transmembrane domains shows higher activity than truncated forms , suggesting that reconstitution in membrane-like environments (liposomes, nanodiscs) may provide more physiologically relevant measurements.

How should recombinant MtgA be stored to maintain optimal activity?

Based on established protocols for the recombinant full-length B. bronchiseptica MtgA protein, the following storage recommendations should be followed to maintain enzymatic activity :

• Short-term storage (up to one week):

  • Store working aliquots at 4°C

  • Avoid repeated freeze-thaw cycles

  • Buffer composition should include Tris/PBS base with 6% trehalose at pH 8.0

• Long-term storage:

  • Store at -20°C/-80°C

  • Add glycerol to a final concentration of 30-50%

  • Aliquot in small volumes to avoid repeated freeze-thaw cycles

  • The commercial preparation recommends reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute the lyophilized protein in deionized sterile water

  • Allow complete dissolution before using in experiments

  • For enzymatic assays, ensure buffer conditions match those used in published protocols

These storage conditions help preserve the integrity of both the catalytic domain and the transmembrane region, which is essential for full enzymatic activity .

How does B. bronchiseptica MtgA compare to homologous enzymes in other Bordetella species?

The MtgA protein from B. bronchiseptica shares significant structural and functional homology with counterparts in other Bordetella species, though with notable differences that may influence substrate specificity and catalytic properties:

• Sequence conservation:

  • High sequence similarity exists between B. bronchiseptica and B. pertussis MtgA

  • Conservation is particularly high in the catalytic domain

  • The transmembrane domains show more variability between species

• Functional differences:

  • B. pertussis is known to release a specific peptidoglycan fragment called tracheal cytotoxin (TCT) which promotes loss of ciliated respiratory epithelium

  • Studies suggest B. pertussis releases a remarkably homogeneous set of peptidoglycan fragments, consisting principally of TCT

  • The potential role of MtgA in generating these specific peptidoglycan fragments requires further investigation

• Evolutionary context:

  • Comparison with more distantly related bacterial glycosyltransferases reveals conservation of key catalytic residues

  • Membrane association appears to be a consistent feature across different bacterial species, with full-length proteins containing transmembrane segments showing higher activity than truncated forms

This comparative analysis provides insight into conserved features essential for enzyme function while highlighting species-specific adaptations that may relate to pathogenesis.

What structural features are essential for substrate binding to MtgA?

Studies on peptidoglycan glycosyltransferases have identified critical structural elements required for substrate recognition and binding:

• Lipid II binding requirements:

  • The D-lactoyl moiety of MurNAc plays a crucial role in substrate recognition

  • Research has shown that lipid II analogs containing D-lactoyl exhibit inhibitory activity against glycosyltransferases even without L-Ala residues, indicating that D-lactoyl is sufficient for binding

  • The peptide component influences binding affinity, with studies showing that pentapeptide and tripeptide derivatives have similar binding affinities, while derivatives with L-Ala residues have intermediate affinity

• Role of transmembrane domains:

  • The transmembrane segment of MtgA and related proteins significantly influences substrate binding

  • Full-length proteins with intact transmembrane domains show stronger interactions with lipid substrates compared to truncated forms

  • This suggests the TM segment may directly participate in substrate recognition, potentially through interaction with the lipid portion of lipid II

• Binding site architecture:

  • Conservation analysis across bacterial species indicates a highly conserved active site architecture

  • Key residues in the catalytic domain coordinate substrate positioning for transglycosylation

These findings are particularly relevant for developing substrate mimics as potential inhibitors and for understanding the natural substrate specificity of B. bronchiseptica MtgA.

How can moenomycin and other inhibitors be used to study MtgA function?

Moenomycin and related inhibitors serve as valuable tools for investigating MtgA structure and function through multiple approaches:

• Mechanistic studies:

  • Moenomycin is a specific inhibitor of glycosyltransferases and has been extensively used to study their properties

  • By mimicking the lipid II substrate, moenomycin provides insight into the substrate binding mechanism

  • Comparing binding affinities of different moenomycin analogs can reveal critical interaction points

• Structural biology applications:

  • Co-crystallization of MtgA with moenomycin or synthetic analogs can elucidate the three-dimensional structure of the active site

  • Research has reported the synthesis of lipid II analogs and their co-crystallization with MtgA proteins

  • These structures reveal how the protein recognizes both natural substrates and inhibitors

• Development of screening tools:

  • Fluorescently labeled moenomycin derivatives have been developed and used to create high-throughput screening assays

  • These assays facilitate the discovery of novel inhibitors with potentially different binding modes

• Substrate binding studies:

  • Competition assays between natural substrates and moenomycin can characterize binding kinetics

  • The observation that moenomycin binds more strongly to full-length proteins with transmembrane domains than to truncated forms provides insight into substrate recognition

These approaches collectively provide complementary information about substrate recognition, catalytic mechanism, and potential inhibition strategies for MtgA.

What is the relationship between MtgA activity and Bordetella virulence factors?

The connection between MtgA activity and Bordetella virulence involves several important mechanisms:

• Peptidoglycan fragment generation:

  • Bordetella pertussis releases a specific peptidoglycan fragment called tracheal cytotoxin (TCT) that promotes loss of ciliated respiratory epithelium

  • These fragments are characterized as 1,6-anhydro-N-acetylmuramic acid-containing disaccharide peptides

  • As a peptidoglycan glycosyltransferase, MtgA likely plays a role in the metabolic processes that generate these fragments

• Immune recognition:

  • T-cell reactivity studies in Bordetella pertussis have shown that Maltose alpha-D-glucosyltransferase (MTHase), which shares functional similarities with MtgA, is among the most reactive antigens recognized by T-cells

  • Approximately 35% of donors showed T-cell responses to MTHase, indicating its potential role in immune recognition during infection

  • This suggests bacterial glycosyltransferases may be important targets of adaptive immunity

• Cell wall integrity:

  • MtgA's role in peptidoglycan synthesis directly affects bacterial cell wall integrity

  • Proper cell wall maintenance is essential for bacterial survival during infection and resistance to host defense mechanisms

Understanding these relationships provides insight into how MtgA activity may contribute to Bordetella pathogenesis and suggests potential therapeutic approaches targeting this enzyme.

Could MtgA be a viable target for novel antimicrobial development?

Several factors suggest MtgA could be a promising target for antimicrobial development:

• Essential cellular function:

  • Peptidoglycan glycosyltransferases play a critical role in bacterial cell wall synthesis

  • Inhibition of these enzymes typically leads to growth arrest or cell lysis

  • The membrane-associated nature of MtgA provides a potentially accessible target for inhibitors

• Existing inhibitor scaffolds:

  • Moenomycin specifically inhibits glycosyltransferases and has been extensively studied

  • Several analogs have been prepared and characterized, including fluorescently labeled derivatives

  • These provide valuable starting points for developing optimized inhibitors

• Substrate mimics as templates:

  • Recent advances in synthesizing lipid IV substrate and higher oligosaccharides of MurNAc-GlcNAc provide templates for inhibitor design

  • These substrate mimics facilitate the study of inhibitor specificity and the design of substrate-based inhibitors

• Immunological considerations:

  • Studies on T-cell responses to Bordetella antigens revealed that MTHase (a related glycosyltransferase) elicits significant responses in 35% of donors

  • This suggests targeting MtgA might synergize with host immune responses

• Research strategies:

  • High-throughput screening assays using fluorescently labeled moenomycin have been developed

  • Structure-based design approaches are feasible given the availability of crystal structures of related enzymes

These factors collectively suggest MtgA is a viable target for antimicrobial development, though successful drug development would require addressing challenges related to selectivity, bioavailability, and resistance mechanisms.

What techniques can elucidate the structure-function relationship of MtgA?

Several advanced techniques can provide detailed insights into MtgA structure-function relationships:

• X-ray crystallography and cryo-EM:

  • Co-crystallization of MtgA with lipid II analogs has been reported for related enzymes

  • For membrane-associated forms, lipidic cubic phase crystallization or cryo-EM may be more suitable

  • These approaches can reveal atomic-level details of substrate binding and catalytic mechanism

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Enables mapping of protein dynamics and ligand-induced conformational changes

  • Particularly useful for identifying regions involved in substrate recognition

  • Can be applied to membrane proteins when combined with appropriate detergent systems

• Site-directed mutagenesis coupled with activity assays:

  • Systematic mutation of conserved residues can identify those critical for catalysis

  • Transmembrane domain modifications can assess the contribution to substrate binding

  • Chimeric proteins with domains from different bacterial species can reveal species-specific adaptations

• Advanced spectroscopic methods:

  • Fluorescence resonance energy transfer (FRET) between labeled enzyme and substrate

  • Single-molecule FRET to monitor conformational changes during catalysis

  • Nuclear magnetic resonance (NMR) for studying protein-ligand interactions in solution

• Molecular dynamics simulations:

  • In silico modeling of enzyme-substrate interactions in membrane environments

  • Prediction of conformational changes upon substrate binding

  • Virtual screening of potential inhibitors

These complementary approaches can provide a comprehensive understanding of how MtgA structure relates to its function in peptidoglycan synthesis.

How can isotope labeling techniques advance our understanding of MtgA activity?

Isotope labeling approaches offer powerful tools for studying MtgA enzymatic activity in vitro and in cellular contexts:

• Mechanistic studies using labeled substrates:

  • Similar to B. pertussis studies where [³H]diaminopimelic acid (DAP) was used to specifically label peptidoglycan

  • Incorporation of isotope-labeled precursors enables tracking of newly synthesized peptidoglycan

  • Can determine reaction rates, processivity, and glycan chain length

• Mass spectrometry applications:

  • ¹⁸O labeling can track the fate of specific hydroxyl groups during transglycosylation

  • ¹⁵N or ¹³C labeling of peptide portions can monitor peptidoglycan remodeling

  • Coupled with high-resolution mass spectrometry, these approaches provide detailed structural information on reaction products

• NMR spectroscopy:

  • ¹³C and ¹⁵N labeling enables solution NMR studies of substrate binding

  • Can provide information on conformational changes upon substrate binding

  • Particularly valuable for studying substrate analogs and inhibitor interactions

• In vivo labeling strategies:

  • D-alanine analogs containing bioorthogonal handles (e.g., azides or alkynes) enable visualization of newly synthesized peptidoglycan

  • Pulse-chase experiments with labeled and unlabeled precursors can monitor peptidoglycan turnover

  • Can be combined with super-resolution microscopy to map peptidoglycan synthesis sites

These isotope labeling approaches provide valuable insights into both the enzymatic mechanism of MtgA and its role in bacterial cell wall synthesis and remodeling.

What are the most promising future research directions for B. bronchiseptica MtgA?

Several high-priority research directions could significantly advance our understanding of B. bronchiseptica MtgA:

• Structural biology:

  • Determination of high-resolution structures of full-length MtgA in membrane-like environments

  • Comparison with homologous enzymes from other bacterial pathogens

  • Elucidation of conformational changes during catalysis

• Role in pathogenesis:

  • Investigation of how MtgA activity relates to the production of immunoactive peptidoglycan fragments

  • Determination of whether MtgA-dependent processes contribute to host immune evasion

  • Assessment of MtgA as a potential vaccine target, considering the observed T-cell reactivity to related glycosyltransferases

• Inhibitor development:

  • Design of species-selective inhibitors based on structural differences

  • Development of peptidomimetics that exploit the D-lactoyl binding requirement

  • Creation of dual-targeting molecules that simultaneously inhibit multiple steps in peptidoglycan synthesis

• Systems biology approaches:

  • Characterization of MtgA interaction networks during infection

  • Identification of regulatory mechanisms controlling MtgA expression

  • Integration of MtgA function with other virulence mechanisms