Recombinant Rhizobium leguminosarum bv. trifolii Adenosylhomocysteinase (ahcY)

What is the genomic context of adenosylhomocysteinase (ahcY) in Rhizobium leguminosarum bv. trifolii?

The ahcY gene is part of the Rhizobium leguminosarum bv. trifolii genome, which has been fully sequenced in several strains. For example, strain WSM1689 has a complete 6,903,379 bp genome containing 6,709 protein-coding genes and 89 RNA-only encoding genes. This multipartite genome contains six distinct replicons: a main chromosome of 4,854,518 bp and five plasmids of varying sizes (667,306, 518,052, 341,391, 262,704, and 259,408 bp) .

Like other Rhizobium species, R. leguminosarum bv. trifolii is classified in the order Rhizobiales of the class Alphaproteobacteria. The genomic organization of ahcY and its flanking regions may provide insights into its regulation and functional relationships with other genes involved in methylation pathways and symbiotic processes.

What expression systems are most effective for producing recombinant R. leguminosarum bv. trifolii AHCY?

While the search results don't specifically address expression systems for R. leguminosarum bv. trifolii AHCY, several systems have been successfully used for recombinant AHCY from other organisms:

• E. coli BL21(DE3) RIL: This bacterial expression system has been effectively used for human AHCY expression, with protocols adapting growth conditions to optimize protein solubility .

• Mammalian cells (HEK293T): This system has been used for human AHCY and may be suitable when post-translational modifications are important .

• Modified E. coli expression conditions: For challenging AHCY constructs, modifications such as:

  • Temperature reduction after induction (37°C to 25-28°C)

  • Addition of benzyl-alcohol (0.1%, 9.65mM) to induce endogenous chaperones

  • IPTG induction at 0.5mM final concentration

For R. leguminosarum bv. trifolii AHCY specifically, an E. coli-based system would likely be most appropriate, with a T7 promoter-based vector such as pET series, and growth conditions optimized to maintain protein solubility.

What are the optimized methods for purifying recombinant AHCY from R. leguminosarum bv. trifolii?

Based on methodologies used for other recombinant AHCY proteins, the following purification strategy would be appropriate:

• Affinity chromatography: Using histidine-tag (His-tag) fusion proteins and nickel or cobalt affinity columns for initial capture .

• For soluble protein:

  • Cell lysis using methods that preserve enzyme activity (sonication in appropriate buffer)

  • Clarification by centrifugation (typically 10,000-20,000 × g for 30 minutes)

  • Binding to affinity resin

  • Washing with buffer containing low imidazole concentrations

  • Elution with higher imidazole concentrations

  • Conventional chromatography steps for further purification

• For insoluble protein:

  • Isolation of inclusion bodies

  • Solubilization using denaturing agents

  • Protein refolding using systems like iFold Protein Refolding System

  • Verification of proper folding through activity assays

Buffer compositions typically include:

• 25-50 mM Tris-HCl or HEPES

• 100-300 mM NaCl

• 10% glycerol

• pH 7.0-7.5

For downstream applications in cell culture, filtration through a 0.2 μm filter is recommended, though some protein loss may occur during this process .

How is AHCY enzyme activity measured in vitro?

Standard AHCY activity assays measure either the hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine or the reverse reaction. Methods include:

Fluorescence-based assay:

• Buffer: 50 mM HEPES, 2 mM MgCl₂, 1 mM EDTA, pH 7.0

• Substrate: Adenosylhomocysteine (commercially available, 10 mM stock in 10 mM HCl)

• Detection of homocysteine production using ThioGlo®3 Fluorescent Thiol Reagent

• Measurement with a fluorescent plate reader in a black 96-well plate format

• Standard curve generated using reduced glutathione

Spectrophotometric assay:

• Monitoring the conversion of SAH to adenosine by coupling to adenosine deaminase

• Following absorbance changes at 265-280 nm

• Calculation of enzymatic rates from the linear portion of the reaction curve

What functional domains and catalytic residues are essential for AHCY activity?

While specific information for R. leguminosarum bv. trifolii AHCY is not provided in the search results, insights from human AHCY and other organisms suggest:

• NAD⁺ binding domain: Essential for the oxidation-reduction steps of the catalytic mechanism

• Substrate binding domain: Undergoes conformational changes during catalysis, with an approximately 18° rotation of the hinge bringing together the cofactor and substrate-binding domains

• Critical residues: Studies of human AHCY mutations have identified several residues crucial for activity:

  • Asp86: The negative charge at this position is essential for catalytic activity. Replacement with glycine causes enzyme inactivation, while restoring the negative charge with glutamic acid restores 70% of wild-type activity

  • Arg49: Important for structural integrity; its replacement with cysteine leads to inappropriate disulfide bond formation and loss of activity

The enzymatic mechanism involves a nucleophilic cascade enabled by redox steps:

• Oxidation of substrate by enzyme-bound NAD⁺

• Cleavage of the oxidized intermediate

• Reduction by NADH to form the final product

How do mutations in specific ahcY residues affect enzyme structure and function?

Research on human AHCY mutations provides a framework for understanding structure-function relationships:

• Arg49Cys mutation effects:

  • Forms intermolecular disulfide bonds

  • Creates enzymatically inactive macromolecular structures

  • Functionality can be partially restored with reducing agents like DTT

  • Alters the oxidation state of bound NAD⁺ cofactor

• Asp86Gly mutation effects:

  • Forms enzymatically inactive aggregates

  • Loss of negative charge critically impacts enzyme function

  • Replacing Gly86 with negatively charged Glu86 restores activity to 70% of wild-type

  • Replacing with positively charged Lys86 or uncharged Leu86 does not improve activity

These findings demonstrate that both proper protein folding and specific charge distributions are essential for AHCY function. When investigating R. leguminosarum bv. trifolii AHCY, similar site-directed mutagenesis approaches could identify critical residues specific to the bacterial enzyme.

What is the role of AHCY in regulating methylation patterns in R. leguminosarum bv. trifolii and how does this affect symbiosis?

While the search results don't directly address AHCY's role in R. leguminosarum bv. trifolii methylation, evidence from other systems suggests important functions:

• Methylation regulation:

  • AHCY removes S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases

  • By controlling SAH levels, AHCY indirectly regulates all cellular methylation reactions

  • In mammalian systems, AHCY can enhance DNA methyltransferase activity, with AHCY overexpression inducing increased DNA methylation

• Potential symbiotic roles:

  • Methylation processes influence gene expression patterns, potentially affecting nodulation genes

  • DNA methylation could regulate host specificity, as R. leguminosarum bv. trifolii strains show host-dependent symbiotic efficiency with different Trifolium species

  • The metabolic state and methylation processes may influence bacterial adaptation to the plant environment

The study of perennial vs. annual host specificity in R. leguminosarum bv. trifolii could be extended to investigate whether AHCY-regulated methylation contributes to host specificity patterns, potentially through epigenetic regulation of key symbiotic genes.

How does AHCY interact with other enzymes in the one-carbon metabolism pathway in R. leguminosarum bv. trifolii?

While not specifically addressed for R. leguminosarum bv. trifolii, AHCY is known to be a key component of the one-carbon metabolism pathway. In this pathway:

• Methionine cycle integration:

  • AHCY hydrolyzes SAH to homocysteine and adenosine

  • Homocysteine can be remethylated to methionine via methionine synthase (MS) or betaine-homocysteine methyltransferase (BHMT)

  • Methionine is converted to S-adenosylmethionine (SAM) via methionine adenosyltransferase (MAT)

  • SAM serves as the methyl donor for various methyltransferases, producing SAH

• Protein interactions:

  • In mammalian systems, AHCY interacts with DNA methyltransferase DNMT1, enhancing its function

  • AHCY has been found to associate with transcription factors at chromatin, suggesting direct involvement in epigenetic regulation

  • Interactions with the circadian transcription factor BMAL1 have been reported, linking methionine metabolism to circadian rhythms

For studying AHCY interactions in R. leguminosarum bv. trifolii, methods like co-immunoprecipitation coupled with mass spectrometry could identify bacterial-specific interaction partners, potentially revealing unique aspects of one-carbon metabolism in this symbiotic bacterium.

What techniques can accurately determine the subcellular localization of AHCY in R. leguminosarum bv. trifolii during different life stages?

While the search results don't specifically address AHCY localization in R. leguminosarum bv. trifolii, several techniques could be applied:

• Immunofluorescence microscopy:

  • Generation of specific antibodies against R. leguminosarum bv. trifolii AHCY

  • Fixation and permeabilization of bacterial cells

  • Fluorescent labeling and confocal microscopy

• Fluorescent protein fusions:

  • Creation of translational fusions with GFP or other fluorescent proteins

  • Expression in R. leguminosarum bv. trifolii under native promoter control

  • Live-cell imaging during free-living growth and symbiotic stages

• Biochemical fractionation:

  • Separation of bacterial subcellular compartments

  • Western blot analysis of fractions with AHCY-specific antibodies

  • Comparison between free-living bacteria and bacteroids from nodules

• Chromatin immunoprecipitation (ChIP):

  • If AHCY associates with DNA or chromatin (as seen in other organisms )

  • Could identify genomic regions where AHCY might be recruited

• In-nodule localization:

  • Root nodule sectioning and immunohistochemistry

  • Could reveal AHCY distribution during symbiotic states

These approaches could determine whether AHCY localization changes during the transition from free-living to symbiotic states, potentially indicating different functional roles during these life stages.

How do environmental factors affect AHCY expression and activity in R. leguminosarum bv. trifolii?

While the search results don't directly address environmental regulation of AHCY in R. leguminosarum bv. trifolii, several factors likely influence its expression and activity:

The observation that metabolically versatile R. leguminosarum bv. trifolii strains are prevalent in nodule populations while metabolically specialized strains show higher symbiotic effectiveness raises interesting questions about how AHCY activity might contribute to this balance between versatility and specialization.

What are the differences in kinetic parameters between recombinant AHCY from R. leguminosarum bv. trifolii compared to other bacterial and eukaryotic AHCYs?

While specific kinetic parameters for R. leguminosarum bv. trifolii AHCY are not provided in the search results, comparative enzymology approaches would include:

• Kinetic parameter determination:

  • Km and Vmax for both forward (SAH hydrolysis) and reverse (SAH synthesis) reactions

  • pH optimum and pH-rate profiles

  • Temperature stability and thermodynamic parameters

  • Effects of potential inhibitors

• Structural basis for kinetic differences:

  • NAD⁺ binding affinity

  • Substrate binding pocket architecture

  • Conformational changes during catalysis

  • Oligomeric state stability

• Comparative analysis framework:

  • Human AHCY is well-characterized, with specific residues (e.g., Arg49, Asp86) known to be critical for activity

  • Comparison with other bacterial enzymes could reveal specific adaptations

  • Mammalian AHCY undergoes conformational changes during catalysis, with an ~18° rotation of the hinge between domains

Such comparative studies could reveal adaptations specific to R. leguminosarum bv. trifolii AHCY that might relate to its symbiotic lifestyle.

How can researchers overcome challenges in expressing functional R. leguminosarum bv. trifolii AHCY in heterologous systems?

Based on experiences with human AHCY expression and general recombinant protein production principles, several strategies can address potential challenges:

• Solubility enhancement:

  • Temperature optimization: Reducing growth temperature after induction (25-28°C)

  • Co-expression with molecular chaperones

  • Addition of 0.1% benzyl alcohol to induce endogenous chaperones

  • Use of solubility-enhancing fusion tags (thioredoxin, SUMO, MBP)

• Addressing protein aggregation:

  • Optimization of buffer conditions (pH, salt concentration, additives)

  • Addition of reducing agents if disulfide bond formation is problematic

  • Screening different detergents for membrane-associated forms

• Refolding from inclusion bodies:

  • Isolation of inclusion bodies using standard protocols

  • Solubilization in denaturing agents

  • Controlled refolding using commercial systems like iFold

  • Activity verification after refolding

• Cofactor incorporation:

  • Ensuring proper NAD⁺ incorporation by supplementing growth media or purification buffers

  • Verification of cofactor binding through spectroscopic methods

• Expression level control:

  • Tuning inducer concentration

  • Using weaker promoters if toxicity is an issue

  • Codon optimization for the expression host

The experience with human AHCY mutations suggests that maintaining proper charge distribution and preventing inappropriate disulfide formation may be particularly important for functional expression .

What computational approaches can predict functional impacts of natural variations in the ahcY gene among different R. leguminosarum bv. trifolii strains?

To analyze natural variations in ahcY among R. leguminosarum bv. trifolii strains, researchers could employ these computational approaches:

• Comparative genomics:

  • Sequence alignment of ahcY genes from multiple strains

  • Identification of conserved vs. variable regions

  • Analysis of selection pressure (dN/dS ratios)

  • Investigation of strain-specific alleles

• Structural modeling:

  • Homology modeling based on crystal structures of AHCY from other organisms

  • Prediction of structural impacts of identified variants

  • Molecular dynamics simulations to assess conformational changes

  • Analysis of effects on substrate binding and catalysis

• Functional impact prediction:

  • Algorithms like SIFT, PolyPhen-2, or PROVEAN to assess mutation impacts

  • Conservation analysis across diverse species

  • Assessment of changes in physicochemical properties

  • Evaluation of potential effects on protein-protein interactions

• Integration with experimental data:

  • Correlation with symbiotic efficiency on different host plants

  • Relationship to metabolic versatility profiles

  • Connection to environmental adaptation patterns

This computational analysis could identify candidate variants for experimental validation and potentially connect AHCY sequence variation to functional differences in symbiotic performance or environmental adaptation.

What are the best controls to include when expressing and characterizing recombinant R. leguminosarum bv. trifolii AHCY?

To ensure reliable results when working with recombinant R. leguminosarum bv. trifolii AHCY, include these controls:

• Expression controls:

  • Empty vector control to assess background expression

  • Known well-expressing protein (e.g., GFP) to validate expression system

  • Negative control lacking inducer to confirm induction specificity

  • Time-course sampling to determine optimal expression timing

• Purification controls:

  • Mock purification from cells with empty vector

  • Use of tagged control protein with known behavior

  • Monitoring of all purification fractions

  • Assessment of batch-to-batch consistency

• Activity assay controls:

  • Commercial AHCY enzyme when available

  • Heat-inactivated enzyme (negative control)

  • Reactions without substrate

  • Standard curves for quantification

  • Assessment of linearity within the working range

• Structural validation controls:

  • Circular dichroism to confirm proper folding

  • Size-exclusion chromatography to verify oligomeric state

  • Native PAGE to assess homogeneity

  • Thermal stability assays

• Mutational controls:

  • Site-directed mutagenesis of known critical residues (based on homologous enzymes)

  • Conservative vs. non-conservative substitutions

  • Charge-reversal mutations to confirm importance of charge distribution

Including these controls will help distinguish true effects from artifacts and ensure the relevance of findings to the native enzyme.

How can researchers effectively isolate and identify R. leguminosarum bv. trifolii strains from root nodules for AHCY studies?

Based on the search results , an efficient method for isolating R. leguminosarum strains from root nodules includes:

• Nodule collection and surface sterilization:

  • Surface sterilize nodules with 2% sodium hypochlorite for 15 minutes

  • Rinse thoroughly with sterile distilled water

  • Process nodules while fresh for highest viability

• Nodule crushing and plating:

  • Use a plexiglass apparatus with machine bolts for simultaneous crushing of multiple nodules

  • Transfer crushed nodule suspension to plates containing selective media

  • Include antibiotic or dye markers for initial screening

• Colony purification and storage:

  • Pick well-isolated colonies onto fresh medium

  • Confirm purity through microscopic examination

  • Prepare glycerol stocks (25-30% glycerol) for long-term storage

• Molecular confirmation:

  • PCR amplification of 16S rRNA gene

  • Species-specific markers like nodD genes

  • Multilocus sequence analysis (MLSA) using housekeeping genes (atpD, recA)

  • BOX-PCR for strain-level differentiation

• AHCY-specific screening:

  • PCR amplification of the ahcY gene

  • Sequencing to identify variants

  • Potential activity-based screening if phenotypic differences are expected

This approach can efficiently process approximately 115 nodules per hour and provides material for subsequent molecular and biochemical analyses of AHCY.

What expression systems are most suitable for producing recombinant R. leguminosarum bv. trifolii AHCY with site-specific mutations?

For producing R. leguminosarum bv. trifolii AHCY variants with specific mutations, consider these expression systems:

• E. coli-based expression:

  • pET system with T7 promoter for high expression levels

  • pQE vectors with 6xHis tags for efficient purification

  • pThioHis or similar thioredoxin fusion systems if solubility is challenging

  • Cold-inducible systems for temperature-sensitive variants

• Site-directed mutagenesis approaches:

  • QuikChange or similar PCR-based methods for single amino acid substitutions

  • Gibson Assembly for more complex modifications

  • Golden Gate cloning for multiple variant generation

• Expression optimization:

  • Temperature reduction after induction (25-28°C)

  • IPTG concentration titration (typically 0.1-1.0 mM)

  • Addition of 0.1% benzyl alcohol to induce chaperones

  • Co-expression with specific chaperones if needed

• Special considerations for specific mutations:

  • For cysteine mutations: inclusion of reducing agents in buffers

  • For charge-altering mutations: buffer pH optimization

  • For folding-sensitive mutations: fusion to solubility-enhancing tags

• Validation approaches:

  • Parallel wild-type expression as control

  • Western blot confirmation with tag-specific antibodies

  • Activity assays to quantify functional impact

  • Structural analysis through circular dichroism or thermal stability assays

These approaches can be tailored based on the specific mutations being studied and their predicted effects on protein stability and function.

How can isothermal titration calorimetry (ITC) be optimized for studying substrate binding to R. leguminosarum bv. trifolii AHCY?

While not specifically mentioned in the search results, ITC is a valuable technique for characterizing enzyme-substrate interactions. For R. leguminosarum bv. trifolii AHCY, consider these optimization strategies:

• Sample preparation:

  • Highly purified protein (>95% purity)

  • Careful buffer matching between protein and substrate solutions

  • Removal of aggregates by centrifugation or filtration

  • Determination of protein concentration by quantitative methods

• Experimental design:

  • Temperature selection based on enzyme stability (typically 25°C)

  • Optimization of protein concentration (typically 10-50 μM)

  • Substrate concentration in syringe 10-20× protein concentration

  • Control experiments: buffer-into-buffer, buffer-into-protein, substrate-into-buffer

• Parameter optimization for AHCY:

  • Inclusion of NAD⁺ in both protein and substrate solutions

  • Testing different buffer systems (HEPES, phosphate)

  • Addition of stabilizing agents if needed

  • Consideration of divalent cations (Mg²⁺) that might affect binding

• Data analysis:

  • Model selection based on binding stoichiometry

  • Determination of binding parameters (Kd, ΔH, ΔS)

  • Global fitting if multiple experiments are performed

  • Correlation with enzymatic activity data

• Comparative studies:

  • Wild-type vs. mutant proteins

  • Different substrates or substrate analogs

  • Inhibitor binding studies

This approach would provide valuable thermodynamic data on substrate recognition by R. leguminosarum bv. trifolii AHCY, complementing kinetic studies.

What methodological approaches can distinguish the functional roles of AHCY in free-living versus symbiotic states of R. leguminosarum bv. trifolii?

To investigate AHCY function across different bacterial life stages, researchers could employ:

• Genetic approaches:

  • Construction of conditional ahcY mutants

  • Promoter replacement with inducible systems

  • Creation of reporter fusions to monitor expression

  • Complementation studies with wild-type and mutant alleles

• Transcriptomic analysis:

  • RNA-seq comparing free-living bacteria vs. bacteroids isolated from nodules

  • qRT-PCR validation of ahcY expression changes

  • Comparison across different host plant species

  • Analysis under various environmental stresses

• Protein-level studies:

  • Western blot quantification of AHCY levels

  • Enzyme activity assays from bacteroids vs. free-living cells

  • Immunolocalization in different bacterial states

  • Proteome-wide analysis of methylated proteins

• Metabolomic approaches:

  • Quantification of SAM/SAH ratio

  • Profiling of one-carbon metabolism intermediates

  • Stable isotope labeling to track methyl group flux

  • Comparison between wild-type and ahcY-modified strains

• Plant-microbe interaction studies:

  • Nodulation assays with wild-type vs. modified strains

  • Host range testing on different Trifolium species

  • Competition experiments in soil or hydroponic systems

  • Microscopic analysis of infection and nodule development

These complementary approaches would provide a comprehensive understanding of how AHCY function differs between free-living and symbiotic states and how these differences impact the plant-microbe relationship.

COG Functional Category Distribution in R. leguminosarum bv. trifolii strain WSM1689

The following table shows the distribution of protein-coding genes associated with general COG functional categories in R. leguminosarum bv. trifolii strain WSM1689, providing context for AHCY's metabolic role:

AHCY would typically be classified in category H (Coenzyme transport and metabolism) or E (Amino acid transport metabolism), which together represent 13.6% of the categorized genes in this strain .

Standard Buffer Compositions for AHCY Purification and Activity Assays

These buffer systems can serve as starting points for R. leguminosarum bv. trifolii AHCY work, with optimization as needed for the specific bacterial enzyme.

Comparative AHCY Activity Data from Mutational Studies

While specific data for R. leguminosarum bv. trifolii AHCY mutations are not available in the search results, human AHCY mutational data provides a valuable reference:

These data highlight the importance of specific amino acid properties (charge, potential for disulfide formation) on AHCY function, providing a framework for designing experiments with R. leguminosarum bv. trifolii AHCY.

Symbiotic Efficiency of R. leguminosarum bv. trifolii Strains with Different Trifolium Species

This comparative data demonstrates the host-dependent symbiotic efficiency relevant to studies of AHCY's potential role in host specificity:

This host-dependent variation in symbiotic performance raises interesting questions about the potential role of methylation processes regulated by AHCY in determining host specificity.

What are common pitfalls in recombinant AHCY expression and how can they be addressed?

Based on experiences with human AHCY expression and general recombinant protein challenges:

For R. leguminosarum bv. trifolii AHCY specifically, adapting these solutions based on the biochemical properties of the bacterial enzyme would be appropriate.

How can researchers address inconsistent results in AHCY activity assays?

To troubleshoot variable AHCY activity measurements:

• Enzyme preparation issues:

  • Ensure consistent protein concentration measurement methods

  • Verify enzyme purity by SDS-PAGE

  • Check for batch-to-batch consistency

  • Use fresh preparations or validate stability during storage

• Assay component quality:

  • Prepare fresh substrate solutions

  • Protect SAH from degradation

  • Use high-quality reagents for detection

  • Verify detector calibration

• Reaction conditions:

  • Control temperature precisely

  • Verify pH of reaction buffer

  • Ensure consistent mixing

  • Control timing of measurements

• Data analysis:

  • Use appropriate standard curves

  • Verify linearity in working range

  • Apply consistent calculation methods

  • Use statistical approaches to identify outliers

• Cofactor considerations:

  • Ensure sufficient NAD⁺ is available

  • Consider adding reducing agents for redox balance

  • Check for interfering compounds in preparations

These approaches can help identify and eliminate sources of variability in AHCY activity measurements.

What strategies can overcome low transformation efficiency when introducing recombinant ahcY into R. leguminosarum bv. trifolii?

For genetic manipulation of R. leguminosarum bv. trifolii:

• Electroporation optimization:

  • Culture cells to early-mid log phase

  • Wash cells extensively to remove salt

  • Use high-quality DNA with appropriate concentration

  • Optimize field strength and pulse duration

  • Allow sufficient recovery time before selection

• Conjugation approaches:

  • Use triparental mating with appropriate helper strains

  • Optimize donor:recipient ratios

  • Use appropriate selective media

  • Ensure plasmid compatibility with host

• DNA preparation considerations:

  • Use unmethylated DNA if restriction systems are present

  • Ensure construct stability in E. coli before transfer

  • Consider plasmid size (smaller constructs usually transform better)

  • Verify promoter compatibility with host

• Recipient strain preparation:

  • Use early to mid-log phase cultures

  • Consider growth conditions that might affect cell wall structure

  • Test multiple wild-type strains which may vary in transformability

• Selection strategies:

  • Use appropriate antibiotic concentrations

  • Allow sufficient time for expression of resistance markers

  • Consider using non-antibiotic selection markers if appropriate