Recombinant Bradyrhizobium japonicum S-adenosylmethionine synthase (metK)

Advanced Research Questions

• What are the optimal conditions for expressing and purifying active recombinant B. japonicum metK?

  Achieving high yields of active recombinant B. japonicum metK requires careful optimization of expression and purification conditions:

  Expression optimization:

  Parameter	Optimal Condition	Notes
  Expression system	Mammalian cells or E. coli BL21(DE3)	Mammalian cells may provide better post-translational modifications
  Induction temperature	16-25°C	Lower temperatures reduce inclusion body formation
  Induction duration	16-24 hours	Longer induction times at lower temperatures improve yield
  Medium supplements	2-5% glycerol, 0.1-0.5% glucose	May improve protein folding and stability
  Induction OD600	0.6-0.8	Optimal cell density before induction

  Purification protocol:

  1. Harvest cells by centrifugation (5,000 × g, 10 min)

  2. Resuspend in buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT)

  3. Lyse cells by sonication or French press

  4. Clarify lysate by centrifugation (15,000 × g, 30 min)

  5. Perform affinity chromatography (Ni-NTA for His-tagged protein)

  6. Further purify by ion exchange chromatography

  7. Final polishing step with size exclusion chromatography

  8. Store at -80°C with 50% glycerol

  Activity maintenance:

  • Add 5-50% glycerol to final preparation

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • Expected purity should be >85% as assessed by SDS-PAGE

• How can the enzymatic activity of recombinant B. japonicum metK be accurately measured?

  Accurate measurement of B. japonicum metK activity involves several complementary approaches:

  Spectrophotometric coupled assay:

  1. Reaction mixture: 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 5 mM ATP, 5 mM L-methionine, purified metK enzyme

  2. Incubate at 30°C (optimal for mesophilic B. japonicum)

  3. Monitor Pi release using malachite green assay or enzyme-coupled system

  4. Calculate initial velocities under different substrate concentrations

  Radiometric assay:

  1. Use 14C-labeled methionine or 35S-labeled methionine

  2. Reaction mixture as above

  3. Terminate reaction at various timepoints using acid precipitation

  4. Quantify labeled SAM formation by scintillation counting

  HPLC-based assay:

  1. Reaction mixture as above

  2. Terminate reactions with perchloric acid

  3. Neutralize with K2CO3

  4. Analyze SAM formation by HPLC with UV detection at 254 nm

  Kinetic parameters should be determined using varying concentrations of substrates (ATP and methionine) to establish Km and Vmax values. For B. japonicum metK, expect sequential kinetic mechanism with random addition of ATP and methionine, similar to what has been observed for M. jannaschii MAT .

• What approaches can be used to study the role of metK in B. japonicum under oxidative stress conditions?

  Studying the role of metK under oxidative stress requires integrated experimental approaches:

  Gene expression analysis:

  1. Culture B. japonicum under prolonged exposure (PE) and fulminant shock (FS) H2O2 conditions as described by Jeon et al. (2011)

  2. Extract RNA at various timepoints

  3. Perform RT-qPCR targeting metK and related genes

  4. Compare with global expression profiles from microarray or RNA-seq data

  Whole-genome expression profiling of B. japonicum under H2O2 stress has shown differential expression of 439 genes under PE and 650 genes under FS conditions . This approach can reveal if metK is among the stress-responsive genes.

  Protein activity assays:

  1. Purify native metK from cells exposed to normal and oxidative stress conditions

  2. Measure enzyme activity using methods described in FAQ #6

  3. Assess changes in kinetic parameters under different redox conditions

  Generation of conditional metK mutants:

  1. Create a metK deletion strain complemented with an inducible metK gene

  2. Assess growth and survival under various H2O2 concentrations with different metK expression levels

  3. Analyze metabolomic changes in SAM and related metabolites

  In vitro oxidative modification analysis:

  1. Expose purified recombinant metK to various oxidants (H2O2, peroxynitrite)

  2. Identify oxidative modifications using mass spectrometry

  3. Correlate modifications with changes in enzyme activity

  This multi-faceted approach can reveal whether metK is a target of oxidative stress and how its activity contributes to stress resistance in B. japonicum.

• What are the key differences between metK from B. japonicum and other rhizobial species?

  Comparative analysis of metK across rhizobial species reveals important differences:

  Sequence and structural comparison:

  Species	Protein Length	Sequence Identity to B. japonicum metK	Key Structural Differences
  B. japonicum	399 aa	100%	Reference structure
  B. diazoefficiens USDA110	399 aa	>98%	Highly conserved, minimal differences
  Sinorhizobium meliloti	395 aa	~75-80%	Different ATP-binding pocket architecture
  Mesorhizobium loti	393 aa	~75-78%	Variations in oligomerization domains
  Rhizobium leguminosarum	396 aa	~75-77%	Different catalytic loop configurations

  Functional differences:

  While primary catalytic function is conserved across rhizobial metK enzymes, differences exist in:

  1. Substrate affinity: Variations in Km values for ATP and methionine

  2. Temperature optima: B. japonicum metK likely has activity profile similar to E. coli MAT at 37°C

  3. pH sensitivity: Different pH optima reflecting adaptation to host plant rhizospheres

  4. Regulation: Different transcriptional and post-translational regulatory mechanisms

  Evolutionary context:

  Phylogenetic analysis places B. japonicum among the BJ group within Bradyrhizobiaceae, closely related to B. japonicum USDA110 . This evolutionary relationship likely influences metK characteristics, with conservation of core catalytic domains but variation in regulatory elements reflecting adaptation to specific plant hosts.

• How can site-directed mutagenesis be used to study the catalytic mechanism of B. japonicum metK?

  Site-directed mutagenesis provides powerful insights into metK catalytic mechanisms:

  Methodology for site-directed mutagenesis studies:

  1. Target selection: Based on sequence alignment with well-characterized MAT enzymes, identify key residues likely involved in:

     • ATP binding

     • Methionine binding

     • Catalysis

     • Oligomerization

  2. Primer design for mutagenesis:

     • Design complementary primers containing desired mutations

     • Include 25-45 nucleotides with mutation in the center

     • Ensure GC content of 40-60%

     • Terminate with G or C bases

  3. PCR-based mutagenesis:

     • Use QuikChange or similar methods with high-fidelity polymerase

     • Digest template DNA with DpnI

     • Transform into competent E. coli

     • Verify mutations by sequencing

  4. Expression and purification:

     • Express wild-type and mutant proteins under identical conditions

     • Purify using affinity chromatography and size exclusion

     • Verify structural integrity using circular dichroism

  5. Kinetic analysis:

     • Determine Km and kcat for wild-type and mutant enzymes

     • Analyze pH dependencies

     • Perform isotope effects studies

  Potential target residues:

  Based on studies of S-adenosylmethionine synthases, key residues likely include:

  • Conserved acidic residues coordinating Mg2+ ions

  • Lysine or arginine residues interacting with ATP phosphates

  • Hydrophobic residues forming the methionine binding pocket

  • Residues involved in transition state stabilization

  Results can be interpreted in the context of the sequential kinetic mechanism, where AdoMet is released first, followed by PPi and Pi .

• What roles does metK play in B. japonicum adaptation to environmental stresses beyond oxidative stress?

  B. japonicum metK likely plays crucial roles in multiple stress responses:

  Temperature stress adaptation:

  • SAM-dependent methylation may modify membrane lipids to maintain fluidity

  • The activation energy for SAM formation in B. japonicum is likely different from thermophilic organisms, affecting temperature adaptation

  Iron limitation response:
  Studies on B. japonicum have identified sophisticated iron acquisition systems including siderophore uptake mechanisms (FsrB, ExsFGH) . SAM-dependent reactions may be involved in:

  • Modification of siderophore structures

  • Regulation of iron transport systems

  • Methylation of regulatory proteins controlling iron homeostasis

  Methodological approach to study these connections:

  1. Create conditional metK mutants or metK overexpression strains

  2. Subject to various stresses (temperature shifts, nutrient limitation, drought)

  3. Analyze growth, survival, and metabolic profiles

  4. Compare transcriptomes and proteomes with wild-type

  5. Measure SAM-dependent modifications (DNA methylation, protein methylation)

  Symbiotic resilience:
  Investigate how metK activity influences:

  • Nodulation under stress conditions

  • Nitrogen fixation efficiency under suboptimal conditions

  • Competition with indigenous soil bacteria

  This research direction is particularly relevant as B. japonicum strains are considered for agricultural applications as biofertilizers , and understanding stress adaptation mechanisms could lead to improved strain performance in field conditions.