Recombinant Putative mycofactocin biosynthesis glycosyltransferase MftF (mftF)

What is mycofactocin (MFT) and what is its biological significance?

Mycofactocin (MFT) is a family of small molecules derived from ribosomally synthesized and post-translationally modified peptides (RiPPs), naturally occurring in many species of Mycobacterium including the pathogenic M. tuberculosis. MFT functions as a redox cofactor involved in alcohol metabolism, particularly in pathways involving nicotinoproteins - enzymes with non-exchangeable bound nicotinamide adenine dinucleotide (NAD) . The name "mycofactocin" derives from three components: "Mycobacterium" (reflecting its prevalence in this genus), "cofactor" (due to its predicted role as an enzymatic cofactor), and "bacteriocin" (because MftC, a critical enzyme in its biosynthesis, is related to enzymes involved in bacteriocin production) .

What is the general structure of mycofactocin?

All mycofactocins share a common precursor called premycofactocin (PMFT), but differ in the glucose-based oligosaccharide chain attached to this core. The foundational structure contains a redox-active α-keto amide moiety that can exist in either oxidized (dione) or reduced (diol) forms, termed mycofactocinone and mycofactocinol respectively . MFT variants are decorated with β-1,4-linked glucose residues, with up to nine glucose units including 2-O-methylglucose modifications in some variants . The nomenclature system designates an MFT with n glucose units as MFT-n, or MFT-nH₂ in its reduced form, while methylated variants are termed methylmycofactocin (MMFT) .

What is the specific role of MftF in mycofactocin biosynthesis?

MftF is the glycosyltransferase responsible for the oligoglycosylation of mycofactocin precursors. It catalyzes the addition of multiple glucose residues to form a β-1,4-glucan chain on the mycofactocin core structure . Research with deletion mutants has demonstrated that MftF is essential not just for the glycosylation process but appears to play a central structural or organizational role in the biosynthetic machinery, as ΔmftF mutants not only lack glycosylated MFT congeners but also show drastically decreased levels of the aglycon precursors PMFT and PMFTH₂ .

What is the complete biosynthetic pathway for mycofactocin production?

The mycofactocin biosynthetic pathway begins with the precursor peptide MftA, which contains a conserved C-terminal Val-Tyr dipeptide core. This process involves several key steps:

• The precursor peptide MftA binds to its chaperone MftB

• The radical SAM enzyme MftC catalyzes oxidative decarboxylation and cyclization of the core peptide

• The peptidase MftE releases the cyclized core to form AHDP (a 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone derivative)

• MftD performs oxidative deamination of AHDP, yielding premycofactocin (PMFT)

• MftF glycosylates PMFT and related intermediates with multiple glucose units

• The final cofactor exists in oxidized and reduced forms

Interestingly, glycosylation by MftF occurs at multiple stages of the pathway, as evidenced by the detection of glycosylated forms of various biosynthetic intermediates including AHDP and GAHDP (glycyl-AHDP) .

How do gene knockouts affect mycofactocin biosynthesis?

Genetic studies using knockout mutants have revealed the following effects:

Complementation studies with reintroduced genes have confirmed these findings, excluding potential polar effects of the genetic manipulations .

What are the structural characteristics of MftF?

The MftF protein from Mycolicibacterium smegmatis (formerly Mycobacterium smegmatis) consists of 470 amino acids and belongs to the glycosyltransferase 2 family (GT2) according to PFAM (PF00535) and CAZy classification . GT2 family enzymes are known for their inverting mechanism of oligoglycoside formation, which aligns with the observed β-configuration of the MFT oligosaccharide chain . The gene encoding MftF is a conserved constituent of MFT biosynthetic loci across different phyla, underscoring the importance of glycosylation in mycofactocin function .

What experimental approaches can be used to verify MftF glycosyltransferase activity in vitro?

To verify MftF glycosyltransferase activity in vitro, researchers can employ several strategies:

• Recombinant protein expression: Express MftF in a heterologous host such as E. coli with appropriate tags for purification

• Enzyme assays: Incubate purified MftF with the substrate (PMFT or related intermediates) and UDP-glucose as the sugar donor

• LC-MS/MS analysis: Detect and characterize the formation of glycosylated products

• Radiochemical assays: Use radiolabeled UDP-glucose to track the transfer of glucose moieties

• Site-directed mutagenesis: Mutate key catalytic residues predicted to be involved in glycosyl transfer to confirm their importance

These approaches should incorporate appropriate controls, including catalytically inactive mutants and assays without the enzyme or substrate .

How does the β-1,4-glucan chain affect mycofactocin function?

The β-1,4-glucan chain attached by MftF appears to be crucial for mycofactocin function, though the exact mechanistic implications are still being explored. Several hypotheses about its role include:

• Modulating the redox potential of the mycofactocin core

• Enhancing solubility and bioavailability within the cell

• Facilitating recognition by partner enzymes, particularly nicotinoprotein dehydrogenases

• Providing protection against degradation

• Enabling proper subcellular localization

The fact that MftF is conserved across mycofactocin-producing species suggests that glycosylation is fundamentally important for cofactor function . Additionally, the observation that MftF mutants show impaired growth on ethanol correlates with the proposed role of mycofactocin in alcohol metabolism .

What metabolomic approaches can be used to study mycofactocin biosynthesis?

Researchers have successfully used a combination of metabolic induction and stable isotope labeling to specifically trace mycofactocin congeners:

• Metabolic induction: Cultivating bacteria in media containing ethanol (10 g L⁻¹) to stimulate mycofactocin production, as MFT is involved in alcohol metabolism

• Stable isotope labeling: Using ¹³C-labeled amino acids (glycine, valine, and tyrosine) that are incorporated into the precursor peptide to identify metabolites derived from the MftA peptide

• MS/MS network analysis: Creating molecular networks based on MS/MS fragmentation patterns to identify related metabolites

• Comparative metabolomics: Comparing wild-type and knockout mutant metabolomes to identify pathway-specific metabolites

These approaches have been pivotal in identifying not only the final mycofactocin products but also various biosynthetic intermediates and their glycosylated derivatives .

How can recombinant MftF be expressed and purified for enzymatic studies?

For recombinant expression and purification of MftF:

• Expression system selection: Choose an appropriate host system, considering that MftF is a mycobacterial protein that may require specific conditions for proper folding. E. coli strains optimized for expression of proteins with rare codons might be suitable.

• Vector design: Engineer an expression vector with appropriate promoters and affinity tags (His-tag, GST-tag, etc.) for efficient purification.

• Expression conditions: Optimize temperature, inducer concentration, and expression duration to maximize soluble protein yield.

• Cell lysis: Use gentle lysis methods to preserve protein activity, potentially incorporating detergents if MftF has membrane association.

• Purification strategy: Employ affinity chromatography followed by size exclusion and/or ion exchange chromatography to obtain pure, active enzyme.

• Activity preservation: Include appropriate stabilizers and glycerol in storage buffers to maintain enzymatic activity.

• Quality control: Assess protein purity by SDS-PAGE and verify identity by mass spectrometry.

For enzymes like MftF that may function within a biosynthetic complex, co-expression with partner proteins might enhance solubility and activity .

What analytical methods are most effective for characterizing glycosylated mycofactocin congeners?

Several complementary analytical approaches have proven effective for characterizing mycofactocin and its congeners:

• High-resolution mass spectrometry (HRMS): For accurate mass determination and initial identification of mycofactocin species

• Tandem mass spectrometry (MS/MS): For structural characterization and confirmation of glycosidic linkages

• Nuclear Magnetic Resonance (NMR) spectroscopy: For definitive structure elucidation, including configuration of glycosidic bonds

• Liquid chromatography (LC): For separation of complex mixtures of mycofactocin congeners prior to analysis

• Ion mobility spectrometry: For additional separation based on molecular shape and size

• Infrared and Raman spectroscopy: For additional structural information and confirmation

These techniques revealed that mycofactocin is decorated with up to nine β-1,4-linked glucose residues, including variants with 2-O-methylglucose modifications .

How can contradictory results in MftF functional studies be reconciled?

When facing contradictory results in MftF functional studies, researchers should consider several factors:

• Genetic background differences: Different strains of mycobacteria might have varying compensatory mechanisms or regulatory networks affecting MftF function.

• Growth conditions: Since mycofactocin production is stimulated by specific carbon sources like ethanol, inconsistent growth conditions might explain divergent results.

• Assay sensitivity: Different detection methods vary in sensitivity, potentially leading to apparent contradictions when certain metabolites are below detection limits in some studies.

• Post-translational modifications: MftF activity might be regulated by modifications not accounted for in all experimental designs.

• Protein-protein interactions: MftF appears to function within a biosynthetic complex, and disruption of these interactions could yield different phenotypes depending on experimental approach.

• Technical considerations: Differences in sample preparation, extraction efficiency, and analytical methods can significantly impact results.

A systematic approach comparing methodologies and carefully controlling experimental variables is crucial for resolving such contradictions .

What is the relationship between MftF activity and ethanol metabolism in mycobacteria?

The relationship between MftF, mycofactocin, and ethanol metabolism appears to be deeply interconnected:

• Mycofactocin formation shows dependence on ethanol presence in growth media, suggesting regulatory connections .

• The ΔmftF mutant shows impaired growth on ethanol, indicating that glycosylated mycofactocin is required for efficient ethanol metabolism .

• Mycofactocin is thought to serve as a redox cofactor for nicotinoprotein dehydrogenases involved in alcohol oxidation, similar to the role of pyrroloquinoline quinone (PQQ) in other bacteria .

• The redox activity of mycofactocin (midpoint potential: -255 mV for the PMFT core) is compatible with its proposed role in electron transfer during alcohol oxidation .

• Metabolomic studies have shown that ethanol significantly induces mycofactocin production, strengthening the functional connection .

These observations collectively suggest that MftF-catalyzed glycosylation is essential for mycofactocin to fulfill its cofactor role in ethanol metabolism pathways .

What bioinformatic approaches can predict substrate specificity in MftF homologs?

To predict substrate specificity in MftF homologs across different species, researchers can employ several bioinformatic approaches:

• Sequence alignment and phylogenetic analysis: Identifying conserved residues and domains across MftF homologs to infer functional conservation.

• Structural modeling: Using homology modeling based on known glycosyltransferase structures to predict the substrate-binding pocket architecture.

• Molecular docking simulations: Virtually screening potential sugar donors and acceptors against the modeled MftF structure.

• Co-evolution analysis: Identifying residues that have co-evolved with substrate specificity determinants.

• Genomic context analysis: Examining the organization of mft gene clusters across species to identify correlations between MftF sequence variations and other pathway components.

• Machine learning approaches: Training algorithms on known glycosyltransferase-substrate pairs to predict specificity in uncharacterized MftF homologs.

These approaches can guide experimental design for functional validation of predicted specificities and potentially reveal novel mycofactocin variants in diverse bacterial species .

What are the promising applications of engineered MftF variants?

Engineered MftF variants could offer several promising applications:

• Designer redox cofactors: Creating mycofactocin variants with tailored redox properties by altering the glycosylation pattern

• Bioorthogonal labeling: Developing MftF variants that can accept modified sugars for selective labeling of mycofactocin-dependent processes

• Biosensors: Constructing mycofactocin-based sensors for alcohols and aldehydes by coupling MftF activity to reporter systems

• Biocatalysis: Exploiting MftF's glycosyltransferase activity for the production of valuable glycosides with defined linkages

• Antimycobacterial targets: Developing inhibitors of MftF as potential therapeutics against pathogenic mycobacteria, including M. tuberculosis

The central role of MftF in mycofactocin biosynthesis and its importance for alcohol metabolism make it an attractive target for both fundamental and applied research .

How might structural studies of MftF advance our understanding of its mechanism?

Structural studies of MftF would significantly advance our understanding in several ways:

• Catalytic mechanism: Revealing the precise arrangement of active site residues involved in glycosyl transfer

• Substrate recognition: Identifying binding pockets for both the sugar donor (likely UDP-glucose) and various acceptor substrates (PMFT, AHDP, etc.)

• Processivity determinants: Understanding how MftF can catalyze the addition of multiple glucose residues in a processive manner

• Protein-protein interactions: Potentially revealing interfaces for interaction with other mycofactocin biosynthetic enzymes

• Conformational changes: Capturing different states of the enzyme during the catalytic cycle

• Structure-guided engineering: Enabling rational design of MftF variants with altered specificity or enhanced activity

Crystal structures or cryo-EM studies of MftF, particularly in complex with substrates or products, would provide invaluable insights for both fundamental understanding and biotechnological applications .