Recombinant Lactobacillus plantarum Putative 3-methyladenine DNA glycosylase (lp_1991)

What is 3-methyladenine DNA glycosylase and what function does lp_1991 serve in L. plantarum?

3-methyladenine DNA glycosylase is an enzyme that helps maintain genomic integrity by excising chemically modified bases from DNA, particularly cytotoxic lesions such as 3-methyladenine (3mA). This enzyme initiates the base excision repair pathway, which is the principal mechanism for eliminating alkylpurines from the genome . In L. plantarum, lp_1991 is annotated as a putative DNA-3-methyladenine glycosylase II based on sequence homology . This gene likely encodes an enzyme that specifically catalyzes the removal of 3mA lesions from DNA, similar to its bacterial counterparts like the Escherichia coli 3-methyladenine DNA glycosylase I (TAG) .

The function of lp_1991 is particularly significant in L. plantarum given this organism's remarkable adaptability to diverse ecological niches, including the human gastrointestinal tract, which may expose it to various DNA-damaging agents . The KEGG database annotations identify lp_1991 as "DNA-3-methyladenine glycosylase II" with the enzyme classification number EC:3.2.2.21 , indicating its predicted role in DNA repair pathways.

How does L. plantarum's genome maintenance relate to its ecological adaptability?

L. plantarum exhibits remarkable flexibility and versatility, inhabiting various environmental niches including the human gastrointestinal (GI) tract . This adaptability is supported by its genome plasticity and robust DNA maintenance systems .

Research using in vivo expression technology has identified 72 L. plantarum genes induced during passage through the GI tract, with several encoding stress-related functions that help the organism survive harsh conditions . The putative 3-methyladenine DNA glycosylase (lp_1991) likely contributes to this survival by repairing DNA damage caused by alkylating agents encountered in diverse environments .

The genome of L. plantarum contains numerous carbohydrate-active enzymes and EPS-producing genes that contribute to its ecological adaptability . Comparative genomic analysis shows high collinearity between L. plantarum YW11 and other L. plantarum strains, with alignment of important functional genes including transporters and regulatory genes . This genomic conservation suggests that DNA repair systems, including lp_1991, may be critical for maintaining genetic integrity across diverse habitats.

Table 1. Genome features of L. plantarum relevant to ecological adaptation

What structural characteristics define 3-methyladenine DNA glycosylases in bacterial systems?

Bacterial 3-methyladenine DNA glycosylases predominantly belong to the helix-hairpin-helix (HhH) superfamily of DNA repair proteins . This structural motif is used by hundreds of repair proteins for binding DNA in a sequence-independent manner . Crystal structures of TAG from Salmonella typhi reveal a globular fold consisting of an α-helical domain containing the HhH motif (helices H and I) and a unique Zn2+-binding domain that tethers the N- and C-termini .

The 3mA binding pocket is located at the interface between these two domains . Despite structural similarities among HhH glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. Unlike other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site, representing the first structural demonstration that conformational relaxation must occur in DNA upon base hydrolysis .

Key structural elements in TAG include:

Based on structural homology, lp_1991 likely shares many of these features, though specific structural studies on the L. plantarum enzyme have not been reported in the provided search results.

What is the molecular mechanism of 3-methyladenine recognition and removal by DNA glycosylases?

The molecular mechanism of 3-methyladenine (3mA) recognition and removal involves several sophisticated steps that have been elucidated primarily through studies of TAG from E. coli and S. typhi .

Crystal structures of S. typhi TAG in complex with abasic DNA and 3mA nucleobase have revealed that TAG engages damaged DNA differently than other HhH glycosylases . The recognition process begins with the enzyme detecting subtle alterations in base structure amid the vast excess of normal DNA . This occurs in two steps: interrogation of the DNA duplex during a processive search and direct read-out of the target base that has been flipped into the active site .

Once the damaged base is identified, TAG uses a single hairpin loop to intercalate the DNA duplex, disrupting base stacking and facilitating access to the damage site . In the base binding pocket, specific interactions occur between the enzyme and the 3mA base. In E. coli TAG, Glu38 and Tyr16 form hydrogen bonds to the Hoogsteen and Watson-Crick faces of 3mA, respectively . The side chains of Trp46 and Trp6 pack against the nucleobase ring, providing additional stabilization .

An invariant glutamine (Gln41) is positioned between 3mA and the abasic site, potentially guiding 3mA into the base binding pocket during base flipping . Notably, TAG lacks the catalytic aspartic acid present in all other HhH glycosylases, suggesting a unique catalytic mechanism .

The excision of 3mA appears to involve ground-state destabilization, where conformational strain in the DNA substrate drives base excision by destabilizing the reaction's ground state . This is supported by the observation that conformational relaxation occurs in the DNA upon base hydrolysis, as evidenced by the position of the abasic ribose in the product complex .

How do the crystal structures of TAG inform our understanding of catalytic mechanisms in 3-methyladenine DNA glycosylases?

The crystal structures of Salmonella typhi TAG alone and in complex with abasic DNA and 3mA provide crucial insights into the catalytic mechanisms of 3-methyladenine DNA glycosylases . These structures reveal several key aspects that differentiate TAG from other DNA glycosylases and illuminate its catalytic strategy.

Unlike other glycosylase-DNA structures where the abasic ribose is fully rotated into the active site, in the TAG complex, the abasic ribose adopts two specific conformations, neither of which is fully flipped into the active site pocket . This unexpected observation represents the first structural demonstration that conformational relaxation occurs in the DNA upon base hydrolysis .

The structures also reveal an extensive network of interactions with bases on both DNA strands, providing a structural rationale for how TAG detects 3mA lesions within DNA . Inside the base binding pocket, a conserved glutamic acid (Glu38) plays a significant role in catalysis . Substitution of this residue with alanine reduces the rate of base excision by ~15-fold, demonstrating its importance in the catalytic mechanism .

Based on these structures, a model for TAG in complex with a 3mA-DNA substrate has been constructed that illustrates the likely mechanism for 3mA excision . The model confirms that the positions of 3mA and abasic DNA in the TAG crystal structure are aligned in biologically relevant orientations . The redirection of the phosphate backbone necessary to link the damage site to the 3mA base illustrates that the DNA structure relaxes after hydrolysis of the glycosylic bond .

These structural insights support a ground-state destabilization mechanism for catalysis, where TAG's enhanced interactions with both the non-lesioned strand and the 3mA base, together with the large distance between the abasic moiety and TAG's active site in the product complex, argue that the 3mA glycosylic bond is strained in the substrate complex .

What is the significance of the conserved glutamic acid residue in the catalytic activity of TAG and its potential homolog in lp_1991?

The conserved glutamic acid residue (Glu38 in E. coli TAG) plays a crucial role in the catalytic activity of 3-methyladenine DNA glycosylases . Structural studies have shown that Glu38 lines the back of the active site pocket and forms hydrogen bonds to the Hoogsteen face of 3mA . This interaction appears critical for proper positioning of the substrate for catalysis.

Mutation studies have demonstrated that substitution of Glu38 with alanine reduces the rate of base excision by approximately 15-fold . This significant reduction in catalytic activity indicates that Glu38 is essential for efficient 3mA excision. Interestingly, this effect is not simply due to the loss of electrostatic interaction between negatively charged Glu38 and positively charged 3mA, as suggested by TAG's weaker binding to positively charged 3,9-dimethyladenine base than to neutral 3mA .

A water-mediated hydrogen bond network extends from Glu38 to the phosphate backbone 3' to the abasic site in the product complex . This network may play a role in stabilizing reaction intermediates during catalysis.

While specific information about the putative homologous residue in lp_1991 is not provided in the search results, sequence alignment and structural modeling would likely identify a conserved glutamic acid residue at an equivalent position. Given the functional importance of this residue in TAG, it would be a prime target for mutagenesis studies in lp_1991 to confirm its role in the catalytic mechanism of the L. plantarum enzyme.

Table 2. Effect of mutations on TAG enzymatic activity

How does the genomic context of lp_1991 in L. plantarum compare to tag genes in other bacteria?

The genomic context of lp_1991 in L. plantarum can provide insights into its regulation and functional relationships. According to the KEGG database, lp_1991 is annotated as DNA-3-methyladenine glycosylase II with the enzyme classification EC:3.2.2.21 . This classification places it in the same functional category as the well-studied 3-methyladenine DNA glycosylases from other bacteria.

In E. coli, the tag gene (encoding 3-methyladenine DNA glycosylase I) has been cloned and overexpressed by placing it under the control of the lac promoter, resulting in a 450-fold increase in enzyme activity compared to wild-type strains . This approach enabled purification of the enzyme to apparent physical homogeneity and determination of its amino acid composition and sequence .

The tag gene in E. coli encodes a protein comprising 187 amino acids with a molecular weight of 21,100 Da . While specific details about the size and composition of the lp_1991 gene product are not provided in the search results, comparative analysis with tag genes from other bacteria could provide insights into its structure and function.

What are optimal approaches for expressing and purifying recombinant lp_1991?

Based on successful strategies used for related enzymes, several approaches can be considered for the expression and purification of recombinant lp_1991:

Expression System Selection:
E. coli is the most commonly used host for recombinant expression of bacterial proteins, as demonstrated for TAG from E. coli . A similar approach placed the tag gene under the control of the lac promoter in pUC8, resulting in a 450-fold increase in 3-methyladenine-DNA glycosylase activity upon IPTG induction . For lp_1991, common E. coli expression strains like BL21(DE3) with plasmids containing T7 or lac promoters would be reasonable starting points.

Optimization Strategies:
For optimal expression of L. plantarum proteins, researchers have used statistical methods to optimize medium composition and culture conditions . The one-factor-at-a-time (OFAT) method can be used to screen carbon sources (glucose, sucrose, maltose, fructose, lactose, and galactose) and nitrogen sources (peptone, tryptone, soytone, yeast extract, beef extract, and malt extract) . Response surface methodology (RSM) can further optimize key components identified through initial screening .

Purification Protocol:
A multi-step purification strategy can be employed:

• Cell lysis using sonication or French press in an appropriate buffer (typically Tris-HCL pH 7.5-8.0 with NaCl)

• Initial clarification by centrifugation at 15,000-20,000 × g

• Affinity chromatography using an N- or C-terminal tag (His6, GST, or MBP)

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing and buffer exchange

A recombinant approach with a His-tag would facilitate purification using immobilized metal affinity chromatography (IMAC), followed by tag removal through protease cleavage if the tag interferes with activity.

Quality Control:
The purified enzyme should be assessed for:

• Purity by SDS-PAGE (expected molecular weight based on amino acid sequence)

• Identity by mass spectrometry

• Activity using specific DNA glycosylase assays (described in section 3.2)

• Structural integrity by circular dichroism spectroscopy

How can the enzymatic activity of recombinant lp_1991 be assayed?

Several complementary approaches can be used to assay the enzymatic activity of recombinant lp_1991:

Substrate Preparation:
DNA substrates containing 3-methyladenine can be prepared by:

• Chemical synthesis of oligonucleotides containing 3mA at specific positions

• Treatment of double-stranded DNA with alkylating agents such as methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

• Enzymatic incorporation of modified bases using DNA polymerases

Base Excision Assays:

• Gel-based assays: Using radiolabeled or fluorescently labeled DNA substrates, the glycosylase activity can be monitored by detecting the appearance of abasic sites after alkaline treatment, which converts abasic sites to strand breaks detectable by gel electrophoresis.

• HPLC-based assays: Quantification of released 3mA bases by high-performance liquid chromatography.

• Fluorescence-based assays: Using DNA substrates with fluorophore-quencher pairs that show increased fluorescence upon base excision.

Kinetic Measurements:
For detailed characterization, determine:

• Michaelis-Menten kinetic parameters (Km, kcat, kcat/Km)

• Substrate specificity by comparing activity against various alkylated bases (3mA, 7mG, 3mG, εA)

• Effects of pH, temperature, and salt concentration on enzyme activity

Comparative Analysis:
Compare the activity of lp_1991 with well-characterized 3-methyladenine DNA glycosylases like E. coli TAG and AlkA under identical conditions to establish functional similarity and unique properties.

Based on studies with TAG, key activity measurements should include the rate of base excision, which can be reduced by mutations in critical residues. For example, substitution of Glu38 with alanine in TAG reduces base excision by ~15-fold, while Gln41Ala mutation reduces activity ~6-fold .

What mutagenesis strategies would be most informative for studying lp_1991 function?

Strategic mutagenesis approaches can provide valuable insights into the function and mechanism of lp_1991:

Sequence-Based Targeted Mutagenesis:
Based on sequence alignment with well-characterized 3-methyladenine DNA glycosylases like TAG, identify conserved residues likely to be important for:

• DNA binding (residues in the helix-hairpin-helix motif)

• Base recognition (residues lining the 3mA binding pocket)

• Catalysis (e.g., the equivalent of Glu38 in E. coli TAG, which reduces activity ~15-fold when mutated to alanine )

• Structural integrity (residues involved in zinc coordination)

These residues can be mutated to alanine to assess their contribution to enzyme function, or to functionally similar amino acids to probe specific interactions.

Domain Swapping:
Create chimeric proteins by swapping domains between lp_1991 and other well-characterized glycosylases to determine:

• Which regions confer substrate specificity

• How the DNA binding domains contribute to damage recognition

• Whether catalytic domains are functionally interchangeable

Systematic Alanine Scanning:
For regions without clear homology to known functional domains, systematic alanine scanning mutagenesis can identify previously unrecognized important residues.

Assessment of Mutant Effects:
For each mutant, assess:

• Protein expression and solubility

• Structural integrity using circular dichroism or thermal stability assays

• DNA binding affinity using electrophoretic mobility shift assays or surface plasmon resonance

• Catalytic activity using base excision assays

• Substrate specificity using various DNA lesions

Table 3. Suggested mutagenesis targets in lp_1991 based on TAG homology

What structural biology techniques would be most appropriate for characterizing lp_1991?

A multi-technique structural biology approach would provide comprehensive characterization of lp_1991:

X-ray Crystallography:
This technique has been successfully applied to determine high-resolution structures of TAG from S. typhi both alone and in complex with abasic DNA and 3mA nucleobase . For lp_1991:

• Screen crystallization conditions using commercial sparse matrix screens

• Optimize promising conditions by varying pH, precipitant concentration, and additives

• For complex structures, co-crystallize lp_1991 with synthetic DNA containing an abasic site and 3mA

• Collect diffraction data at synchrotron sources for highest resolution

• Solve the structure by molecular replacement using TAG structures as search models

NMR Spectroscopy:
Complementary to crystallography, NMR can provide insights into protein dynamics and ligand interactions in solution:

• Prepare isotopically labeled protein (15N, 13C) for multidimensional NMR

• Perform backbone and side-chain assignments

• Study ligand binding using chemical shift perturbation experiments

• Investigate conformational changes upon substrate binding

Cryo-Electron Microscopy:
For studying larger complexes or conformational states that may be difficult to crystallize:

• Prepare samples of lp_1991 bound to longer DNA substrates

• Collect images using state-of-the-art cryo-EM equipment

• Perform 3D reconstruction to visualize the enzyme-DNA complex

• Collect SAXS data on lp_1991 alone and in complex with DNA

• Generate low-resolution envelope models

• Compare with atomic models from crystallography or homology modeling

Computational Approaches:

• Homology modeling based on TAG structures to predict lp_1991 structure

• Molecular dynamics simulations to study protein-DNA interactions and conformational changes

• Docking studies to predict binding modes of various substrates

The combination of these techniques would provide a comprehensive structural characterization of lp_1991, from atomic-level details of the active site to large-scale conformational changes upon substrate binding.

How does L. plantarum lp_1991 compare to other bacterial 3-methyladenine DNA glycosylases?

Based on functional annotation and genomic analysis, lp_1991 can be compared with other bacterial 3-methyladenine DNA glycosylases:

Sequence and Structural Comparison:
Bacterial 3-methyladenine DNA glycosylases can be classified into different families, with varying substrate specificities despite structural similarities . TAG and MagIII are highly specific for 3mA, whereas AlkA can excise 3mA, 7mG, and other modified bases . Based on KEGG annotation, lp_1991 is classified as DNA-3-methyladenine glycosylase II (EC:3.2.2.21) , suggesting functional similarity to AlkA rather than TAG.

Substrate Specificity:
Different bacterial 3-methyladenine DNA glycosylases show varying substrate preferences. The table below illustrates the specificity patterns across different enzymes:

Table 4. Substrate specificity of bacterial 3-methyladenine DNA glycosylases

This comparison (adapted from reference ) shows that while all these enzymes can remove 3-methyladenine, they differ in their ability to process other alkylated bases. The specific substrate range of lp_1991 would need to be determined experimentally, but its classification suggests a broader specificity similar to AlkA.

Structural Family:
Most bacterial alkylpurine DNA glycosylases, including TAG, AlkA, and MagIII, belong to the helix-hairpin-helix (HhH) superfamily . This structural motif is used by hundreds of repair proteins for binding DNA in a sequence-independent manner . Based on its annotation, lp_1991 likely also belongs to this superfamily, but specific structural studies would be needed to confirm this classification.

Evolutionary Relationships:
Comparative genomic analysis of L. plantarum shows high collinearity between different strains , suggesting conservation of important functional genes including DNA repair enzymes. Phylogenetic analysis of 3-methyladenine DNA glycosylases across bacterial species could provide insights into the evolutionary history and functional diversification of these enzymes.

How might the function of lp_1991 relate to L. plantarum's probiotic properties?

The function of lp_1991 as a putative 3-methyladenine DNA glycosylase may contribute significantly to L. plantarum's probiotic properties through several mechanisms:

Stress Resistance and Survival:
L. plantarum is known for its ability to survive passage through the human gastrointestinal tract in an active form . The GI environment exposes bacteria to various stressors, including oxidative stress and DNA-damaging agents . Efficient DNA repair systems, including lp_1991, likely contribute to L. plantarum's survival in these harsh conditions by maintaining genomic integrity.

In vivo expression technology studies have identified 72 L. plantarum genes that are induced during passage through the GI tract, including four genes involved in stress-related functions . While lp_1991 was not specifically mentioned among these genes in the search results, other DNA repair enzymes may be upregulated in response to GI tract conditions.

Genomic Stability and Adaptation:
L. plantarum's genome plasticity enables its adaptation to diverse ecological niches . Proper DNA repair mechanisms are essential for maintaining this adaptive capacity by ensuring that beneficial mutations are preserved while deleterious DNA damage is repaired. The putative 3-methyladenine DNA glycosylase encoded by lp_1991 would contribute to genomic stability by removing cytotoxic 3mA lesions that could otherwise lead to cell death .

Interaction with Host Immune System:
Probiotics like L. plantarum can modulate the host immune system . DNA repair enzymes help maintain the proper bacterial phenotype, ensuring consistent interaction with host immune cells. Additionally, bacterial DNA itself can interact with host pattern recognition receptors, and proper DNA repair may influence these interactions.

Competitive Exclusion of Pathogens:
L. plantarum produces diverse and potent bacteriocins, which are antimicrobial peptides that can inhibit pathogenic bacteria . Genomic stability, supported by DNA repair enzymes like lp_1991, ensures consistent production of these beneficial compounds. Research has shown that L. plantarum administration can attenuate inflammation of colitis by altering the gut microbiota composition, including increasing the abundance of beneficial Bacteroides species .

The relationship between DNA repair functions and probiotic properties represents an interesting area for future research, potentially linking molecular mechanisms of genome maintenance to broader ecological and health-promoting roles of L. plantarum.

What experimental approaches could validate the predicted function of lp_1991 in vivo?

To validate the predicted function of lp_1991 as a 3-methyladenine DNA glycosylase in vivo, several complementary experimental approaches can be employed:

Gene Deletion and Complementation:

• Generate an lp_1991 knockout strain using CRISPR-Cas9 or traditional homologous recombination approaches

• Assess sensitivity of the knockout strain to DNA alkylating agents (e.g., MMS, MNNG)

• Complement the knockout with plasmid-expressed lp_1991 to restore resistance

• Perform cross-complementation with known 3-methyladenine DNA glycosylases from other species

DNA Damage Accumulation Analysis:

• Expose wild-type and lp_1991 knockout strains to alkylating agents

• Quantify 3-methyladenine lesions in genomic DNA using mass spectrometry or immunodetection

• Monitor DNA damage response markers in both strains

• Track mutation rates and spectra in the presence and absence of lp_1991

In vivo Expression Analysis:

• Use reporter gene fusions to monitor lp_1991 expression under various conditions

• Determine if expression is induced by DNA damaging agents

• Assess expression during growth in different environments, including GI tract conditions

• Identify transcription factors regulating lp_1991 expression

Functional Genomics Approaches:

• Perform RNA-seq on wild-type and lp_1991 knockout strains to identify compensatory pathways

• Use ChIP-seq to identify genomic regions where the enzyme binds in vivo

• Conduct transposon mutagenesis to identify synthetic lethal interactions with lp_1991

Ecological Fitness Assessment:

• Compare survival of wild-type and knockout strains during passage through the mouse GI tract, similar to previous studies with L. plantarum

• Evaluate competitive fitness in mixed cultures under various stress conditions

• Assess biofilm formation capacity and stress resistance

Table 5. Experimental approaches for validating lp_1991 function in vivo

These approaches would provide comprehensive validation of the predicted function while also potentially revealing novel aspects of lp_1991's role in L. plantarum biology.

How could computational approaches aid in predicting substrate specificity of lp_1991?

Computational approaches offer powerful tools for predicting the substrate specificity of lp_1991 before experimental validation:

Sequence-Based Analysis:

• Multiple Sequence Alignment (MSA): Align lp_1991 with characterized 3-methyladenine DNA glycosylases (TAG, AlkA, MagIII) to identify conserved residues in substrate-binding regions .

• Conservation Analysis: Calculate conservation scores for each position to identify functionally important residues.

• Motif Detection: Identify sequence motifs associated with specific substrate preferences in known glycosylases.

• Machine Learning Classification: Train algorithms on sequences with known specificities to predict lp_1991's substrate range.

Structure-Based Modeling:

• Homology Modeling: Generate a 3D model of lp_1991 based on crystal structures of bacterial 3-methyladenine DNA glycosylases .

• Active Site Analysis: Characterize the shape, electrostatic properties, and hydrophobicity of the predicted binding pocket.

• Molecular Docking: Dock various potential substrates (3mA, 7mG, εA) into the modeled binding site to predict binding affinities and orientations.

• Molecular Dynamics Simulations: Simulate the dynamics of enzyme-substrate complexes to assess stability and identify key interactions.

Network-Based Approaches:

• Genomic Context Analysis: Examine genes adjacent to lp_1991 for functional associations with specific DNA repair pathways.

• Protein-Protein Interaction Prediction: Identify potential interaction partners that might influence substrate specificity.

• Phylogenetic Profiling: Compare the presence/absence patterns of lp_1991 and other genes across species to infer functional relationships.

Integrated Analysis Pipeline:

• Generate initial predictions using sequence analysis

• Refine with structure-based modeling

• Validate predictions using in silico mutagenesis

• Rank potential substrates by predicted binding affinity and catalytic efficiency

• Design targeted experiments to test computational predictions

This multi-layered computational approach would provide testable hypotheses about lp_1991's substrate specificity, guiding subsequent experimental validation and potentially revealing unique features of this enzyme compared to other bacterial 3-methyladenine DNA glycosylases.