Recombinant Desulfovibrio vulgaris Formamidopyrimidine-DNA glycosylase (mutM)

What is Formamidopyrimidine-DNA glycosylase (MutM) and what is its function?

Formamidopyrimidine-DNA glycosylase (MutM) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The enzyme plays a critical role in the base excision repair pathway, specifically targeting and removing oxidative DNA lesions such as 8-oxoguanine. In Desulfovibrio vulgaris, MutM appears to function primarily as a protective mechanism against oxidative stress when this anaerobic bacterium is exposed to oxygen .

The MutM protein demonstrates 8-oxoguanine-DNA glycosylase activity comparable to that found in Escherichia coli MutM, suggesting conservation of this fundamental repair mechanism across bacterial species despite their different ecological niches .

What is the structural composition of Desulfovibrio vulgaris MutM?

The MutM protein from Desulfovibrio vulgaris (Miyazaki F) consists of 336 amino acids. Notably, when compared to MutM proteins from other bacteria, it contains an additional insert of 64 amino acids, which represents a significant structural difference .

While specific structural details of D. vulgaris MutM are not fully characterized in the provided research, studies of Formamidopyrimidine-DNA glycosylase (Fpg, a functionally equivalent enzyme) from E. coli show that the enzyme is a bilobal protein with a wide, positively charged DNA-binding groove. The structure includes a conserved zinc finger and a helix-two-turn-helix motif that participate in DNA binding .

How is the MutM gene expressed in Desulfovibrio vulgaris?

In Desulfovibrio vulgaris (Miyazaki F), both mRNA and protein levels of MutM are naturally low under standard anaerobic growth conditions. This suggests that MutM expression may be induced primarily in response to oxidative stress. The limited expression under normal conditions is consistent with the anaerobic lifestyle of D. vulgaris, where oxidative damage would be minimal in the absence of oxygen .

Research indicates that MutM likely serves as a protective mechanism against the mutagenic effects of oxygen when oxidative stress exceeds the capacity of the organism's primary defense systems against oxygen toxicity .

What are the key structural features of MutM that facilitate DNA lesion recognition?

MutM/Fpg employs several structural elements for DNA damage recognition and processing. Based on crystallographic studies of E. coli Fpg (which shares functional similarity with MutM), the enzyme contains absolutely conserved residues including Lys-56, His-70, Asn-168, and Arg-258 that form hydrogen bonds to the phosphodiester backbone of DNA .

When MutM binds to damaged DNA, it causes a sharp kink at the lesion site. Three key residues (Met-73, Arg-109, and Phe-110) are inserted into the DNA helix, filling the void created by nucleotide eversion—the process where the damaged base is flipped out of the DNA helix. A deep hydrophobic pocket in the active site accommodates this everted base, allowing the enzyme to perform its catalytic function .

How does the additional 64 amino acid insert in D. vulgaris MutM affect its function compared to other bacterial MutM proteins?

The 64 amino acid insert present in Desulfovibrio vulgaris MutM represents a significant structural difference compared to MutM proteins from other bacteria . While the specific functional implications of this insert are not fully characterized in the provided research, it raises important questions about potential adaptive modifications.

Despite this structural difference, kinetic analysis has shown that purified His-tagged D. vulgaris MutM demonstrates 8-oxoguanine-DNA glycosylase activity comparable to that of E. coli MutM . This suggests that the core catalytic function is preserved despite the additional amino acid sequence.

Further structural and functional studies would be needed to determine whether this insert provides any specialized adaptation for the anaerobic lifestyle of D. vulgaris or offers enhanced protection during occasional oxygen exposure.

How is MutM regulated under oxidative stress conditions in anaerobic bacteria?

The regulation of MutM in other bacteria, such as E. coli, shows that MutM is controlled by sigma factor 32, which is typically associated with heat shock and stress responses . Interestingly, in E. coli with a tgt gene deletion (affecting tRNA modification), MutM showed increased mRNA levels but decreased protein levels, suggesting complex post-transcriptional regulation mechanisms .

This discrepancy between transcriptional and translational regulation highlights the sophisticated regulatory networks controlling DNA repair enzyme expression and emphasizes the importance of studying both mRNA and protein levels when characterizing enzyme regulation.

What are the optimal methods for cloning and expressing recombinant D. vulgaris MutM?

Based on successful approaches documented in the literature, the following methodology can be employed for cloning and expressing D. vulgaris MutM:

• Gene isolation: The MutM gene can be isolated from Desulfovibrio vulgaris (Miyazaki F) genomic DNA. In previous work, a 5.9-kb DNA fragment was isolated using XhoI and PvuII restriction enzymes, which contained the MutM gene along with other open reading frames .

• Expression system: Construct an expression system under the control of the T7 promoter in E. coli. This approach has proven effective for obtaining functional MutM protein .

• Purification strategy: A typical purification protocol involves:

  • Tandem anion/cation exchange chromatography using 5 ml HiTrap Q HP and 5ml HiTrap Sp HP columns

  • Running buffer containing 20 mM Tris-HCl (pH 7.4), 125 mM NaCl, 2 mM βME, 10% glycerol

  • Elution with a gradient buffer containing 20 mM Tris-HCl (pH 7.4), 1 M NaCl, 2 mM βME, 10% glycerol

  • Size exclusion chromatography using Superdex 200 pg, XK 16/60 equilibrated in 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 2 mM βME, 10% glycerol

  • Concentration to approximately 8 mg/ml for storage

• Verification: Confirm protein identity using mass spectrometry and assess activity through enzymatic assays targeting 8-oxoguanine excision .

What techniques are most effective for structural determination of MutM-DNA complexes?

Based on successful structural studies of similar DNA glycosylases, the following techniques have proven effective:

• Crystallization of MutM-DNA complexes: Generate a covalently cross-linked complex by trapping the Schiff base intermediate formed during the reaction between MutM and DNA using reduction with sodium borohydride .

• X-ray crystallography: Data collection and processing using the XDS package for indexing, merging, and scaling the diffraction data .

• Structure determination by molecular replacement:

  • Use previously solved structures (e.g., MutM from E. coli, PDB ID: 1K82) as search models

  • For new structures without close homologs, models can be generated using tools like Phyre2

  • Use Phaser for molecular replacement calculations

  • For multi-domain proteins like MutM, better results may be achieved by separating flexible domains (CTD and NTD) and finding individual solutions independently

• Model building and refinement:

  • Manual rebuilding of flexible linkers in Coot

  • Refinement using Phenix Refine

  • Visualization using PyMOL for structural analysis

What assays can be used to measure MutM activity and regulation in anaerobic bacteria?

Several approaches can be employed to study MutM activity and regulation:

• Glycosylase activity assays: Measure the 8-oxoguanine-DNA glycosylase activity using substrates containing 8-oxoG lesions. Kinetic analysis can quantify enzyme efficiency and allow comparison with MutM from other organisms .

• Expression analysis:

  • qRT-PCR to measure mRNA levels

  • Western blotting to quantify protein expression

  • Use of reporter gene constructs (e.g., lacZ fusions) to monitor promoter activity under different conditions

• Oxidative stress induction: Expose anaerobic cultures to controlled levels of oxygen or other oxidative agents to study MutM induction. This can be paired with time-course sampling to track expression dynamics .

• Genetic approaches:

  • Gene knockout studies to assess phenotypic consequences of MutM deficiency

  • Complementation assays to confirm gene function

  • P1 transduction can be used to move mutations between strains for comparative studies

• Protein-DNA interaction assays: Techniques such as electrophoretic mobility shift assays (EMSAs) or fluorescence anisotropy to study the binding of MutM to damaged DNA substrates.

How does the DNA binding mechanism of MutM compare between aerobic and anaerobic bacteria?

The DNA binding mechanism of MutM/Fpg appears to be conserved across different bacterial species regardless of their oxygen requirements. In E. coli (a facultative anaerobe), crystal structure analysis reveals that Fpg is a bilobal protein with a wide, positively charged DNA-binding groove that includes a conserved zinc finger and a helix-two-turn-helix motif .

When binding to damaged DNA, the enzyme causes a sharp kink at the lesion site. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA. Three residues (Met-73, Arg-109, and Phe-110) are inserted into the DNA helix to fill the void created when the damaged nucleotide is everted from the DNA helix .

While detailed structural comparisons between MutM from strictly anaerobic bacteria (like D. vulgaris) and aerobic/facultative bacteria are not fully characterized in the provided research, the functional conservation suggests similar binding mechanisms. The additional 64 amino acid insert in D. vulgaris MutM may influence DNA binding in ways that warrant further investigation, potentially providing adaptations specific to the anaerobic lifestyle .

What conformational changes occur in MutM upon DNA binding and catalysis?

Upon binding to damaged DNA, MutM undergoes significant conformational changes that are essential for its catalytic function. Based on structural studies of related DNA glycosylases, the following conformational changes likely occur:

• Domain movement: The bilobal structure of MutM allows for dynamic movement between domains upon DNA binding .

• DNA distortion: MutM binding causes a sharp kink in the DNA at the lesion site, which is necessary for proper positioning of the damaged base .

• Base eversion: During recognition and catalysis, the damaged nucleotide is flipped out (everted) from the DNA helix into a hydrophobic pocket in the enzyme's active site .

• Void filling: As the damaged base is everted, specific residues (Met-73, Arg-109, and Phe-110 in E. coli Fpg) are inserted into the DNA helix to fill the void and stabilize the distorted DNA structure .

• Catalytic positioning: The everted base is positioned in the active site pocket for optimal catalytic attack, enabling the glycosylase activity that cleaves the N-glycosidic bond between the damaged base and the deoxyribose .

These conformational changes represent a coordinated molecular mechanism that allows MutM to efficiently recognize, access, and repair oxidative DNA damage.

How do the kinetic parameters of D. vulgaris MutM compare with those from other bacteria?

The kinetic analysis of purified His-tagged MutM from Desulfovibrio vulgaris shows that it possesses 8-oxoguanine-DNA glycosylase activity comparable to that of MutM from Escherichia coli . This suggests that despite the structural differences, particularly the additional 64 amino acid insert in D. vulgaris MutM, the core catalytic efficiency is maintained.

While specific kinetic parameters (Km, kcat, kcat/Km) are not explicitly provided in the available research data, the functional comparison indicates similar catalytic capabilities between these evolutionarily distinct bacteria .

This conservation of enzymatic activity across bacterial species that occupy different ecological niches (anaerobic sulfate-reducing bacteria versus facultative anaerobes) highlights the fundamental importance of this DNA repair mechanism throughout bacterial evolution.

What evolutionary adaptations are observed in MutM proteins from different bacterial species?

MutM proteins show several evolutionary adaptations across different bacterial species:

• Sequence conservation and divergence: While the core functional domains of MutM are conserved across bacterial species, there are significant sequence variations, such as the additional 64 amino acid insert found in Desulfovibrio vulgaris MutM .

• Expression regulation: In anaerobic bacteria like D. vulgaris, MutM is expressed at low levels under normal conditions but may be induced under oxidative stress. This contrasts with aerobic bacteria where MutM might be more constitutively expressed due to regular exposure to oxygen .

• Regulatory control: In E. coli, MutM is controlled by sigma factor 32, which is typically associated with stress responses. This regulatory mechanism may vary between bacterial species depending on their environmental niche and exposure to DNA-damaging agents .

• Structural adaptations: While the catalytic mechanism appears conserved, structural variations like the additional insert in D. vulgaris MutM may provide specialized functions or stability adaptations relevant to the organism's lifestyle .

These adaptations reflect the diverse environmental challenges faced by different bacterial species while maintaining the essential DNA repair function necessary for genomic integrity.

How does the role of MutM differ between aerobic and anaerobic bacteria?

The role of MutM shows both similarities and differences between aerobic and anaerobic bacteria:

• Core function: In both aerobic and anaerobic bacteria, MutM functions as a DNA repair enzyme that excises oxidized purines, particularly 8-oxoguanine, from damaged DNA .

• Expression patterns:

  • In anaerobic bacteria like Desulfovibrio vulgaris, MutM is expressed at low levels under normal anaerobic conditions, with induction potentially occurring during oxidative stress .

  • In aerobic or facultative anaerobic bacteria, MutM may be more constitutively expressed due to regular exposure to oxygen and resulting DNA damage.

• Protective role:

  • In anaerobic bacteria, MutM appears to serve as an emergency response system, activated when oxygen exposure exceeds the capacity of primary oxygen defense systems .

  • In aerobic bacteria, MutM functions as part of the routine DNA maintenance system, continuously repairing oxidative damage that occurs during normal aerobic metabolism.

• Regulatory integration: The regulatory networks controlling MutM expression likely differ between aerobic and anaerobic bacteria, reflecting their different lifestyles and exposure to oxidative stress. In E. coli, MutM is controlled by sigma factor 32, which regulates stress responses .

These differences highlight how a conserved DNA repair mechanism has been integrated into different bacterial lifestyles through regulatory and potentially structural adaptations.

What are the most promising approaches for studying the function of the unique 64 amino acid insert in D. vulgaris MutM?

To elucidate the function of the unique 64 amino acid insert in Desulfovibrio vulgaris MutM, several approaches could be employed:

• Deletion mutagenesis: Generate recombinant D. vulgaris MutM variants with the 64 amino acid insert deleted and compare their enzymatic activities, DNA binding affinities, and structural stability with the wild-type protein.

• Domain swapping: Create chimeric proteins by swapping the insert region between D. vulgaris MutM and MutM from other bacteria to assess the impact on function and specificity.

• Structural determination: Solve the crystal structure of D. vulgaris MutM both alone and in complex with DNA, focusing on the position and interactions of the insert region. Compare this with structures of MutM/Fpg from other bacteria .

• Molecular dynamics simulations: Conduct computational analyses to predict how the insert affects protein dynamics, particularly during DNA binding and catalysis.

• Expression under varying conditions: Examine how the expression and activity of wild-type and insert-deleted MutM variants respond to different levels of oxidative stress in D. vulgaris.

• Evolutionary analysis: Conduct comparative genomics to identify related anaerobic bacteria with similar inserts and analyze the evolutionary conservation patterns of this region.

These approaches would provide valuable insights into whether this unique insert represents a specialized adaptation for the anaerobic lifestyle of D. vulgaris or serves another function in DNA damage recognition and repair.

How might understanding MutM function lead to new applications in biotechnology or medicine?

Understanding MutM function has several potential applications:

• Biomarkers for oxidative stress: Developing assays based on MutM activity or expression could provide sensitive measures of oxidative stress in biological systems.

• Cancer research: Since oxidative DNA damage is implicated in carcinogenesis, understanding MutM function could contribute to cancer prevention or treatment strategies.

• Anaerobic biotechnology: Insights from D. vulgaris MutM could improve genetic stability in industrial anaerobic bioprocesses, such as biofuel production or waste treatment.

• Antimicrobial development: Differences between bacterial and human DNA repair systems could potentially be exploited for selective antimicrobial strategies.

• Synthetic biology tools: Engineered MutM variants could be developed as tools for specific DNA modifications or as components in synthetic genetic circuits responding to oxidative stress.

• Environmental monitoring: MutM-based biosensors could detect environmental genotoxins or serve as indicators of ecosystem health.

These applications highlight the broader significance of basic research on DNA repair enzymes like MutM across different bacterial species.