Recombinant Corynebacterium glutamicum Adenosylhomocysteinase (ahcY)

What is Adenosylhomocysteinase (ahcY) and what is its function in C. glutamicum?

Adenosylhomocysteinase (ahcY), also referred to as S-adenosylhomocysteine hydrolase (SAHase), is a crucial enzyme in the metabolism of sulfur-containing amino acids in Corynebacterium glutamicum. This enzyme catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine, playing a critical role in the regulation of methylation reactions in the cell.

The enzyme serves as a key component in the methyl cycle, where S-adenosylmethionine (SAM) is utilized as a methyl donor in various cellular processes. After methyl group transfer, SAM is converted to SAH, which is a potent inhibitor of SAM-dependent methyltransferases. The efficient removal of SAH by adenosylhomocysteinase is therefore essential for maintaining proper cellular methylation reactions and metabolic homeostasis in C. glutamicum .

What are the standardized cloning procedures for recombinant C. glutamicum adenosylhomocysteinase?

The established protocol for cloning the adenosylhomocysteinase gene from C. glutamicum involves:

• Gene identification and isolation: The sahase gene is identified and isolated from Corynebacterium glutamicum strain ATCC 13032. This gene consists of an open reading frame of 1,434 nucleotides encoding 478 amino acids .

• Vector selection and preparation: The pET28a+ expression vector is commonly used, which provides an IPTG-inducible promoter system and introduces an N-terminal His-tag for simplified purification.

• Cloning strategy:

  • Design primers to amplify the complete 1,434 bp sahase gene fragment

  • Include appropriate restriction sites for directional cloning

  • Perform PCR amplification using high-fidelity DNA polymerase

  • Digest both the PCR product and pET28a+ vector with corresponding restriction enzymes

  • Ligate the digested gene into the prepared vector

  • Transform the ligated product into a cloning strain of E. coli

• Verification: Confirm correct insertion through restriction analysis and DNA sequencing to ensure the gene is in-frame with the His-tag and free of mutations .

Why is C. glutamicum preferred as an expression system for recombinant proteins?

C. glutamicum offers several significant advantages as an expression system for recombinant proteins, particularly for research applications:

• Safety profile: C. glutamicum is classified as a GRAS (Generally Recognized As Safe) organism, making it suitable for various research applications without special biosafety concerns .

• Low protease activity: The bacterium exhibits naturally low protease activity in culture supernatants, which helps maintain the integrity of expressed recombinant proteins and is particularly beneficial for protease-sensitive proteins .

• Absence of endotoxins: As a gram-positive bacterium, C. glutamicum lacks lipopolysaccharides (endotoxins) that are present in gram-negative bacteria like E. coli. This eliminates the need for endotoxin removal steps during protein purification .

• Secretion capacity: C. glutamicum can efficiently secrete proteins into the culture medium, which can simplify downstream processing and purification procedures .

• Metabolic adaptability: The bacterium has well-characterized metabolic pathways that can be engineered for optimal protein production and cofactor regeneration .

These characteristics make C. glutamicum particularly suitable for expressing adenosylhomocysteinase and other recombinant proteins for research applications.

What expression vectors are suitable for adenosylhomocysteinase production in C. glutamicum?

For successful expression of adenosylhomocysteinase in C. glutamicum, several vector systems can be employed:

• E. coli-C. glutamicum shuttle vectors: These allow for convenient cloning in E. coli followed by expression in C. glutamicum. For adenosylhomocysteinase expression, vectors containing the following components are recommended:

  • Promoter options:

    • IPTG-inducible promoters like P_lacUV5 for controlled expression

    • Auto-inducible promoters like the engineered SigB-dependent cg3141 promoter (P_4-N14) that activates during transition to stationary phase

    • Constitutive promoters derived from C. glutamicum genome (P_sodA, P_tuf) for constant expression

  • Selection markers: Kanamycin or chloramphenicol resistance genes that function in both E. coli and C. glutamicum

  • Origin of replication: Compatible with both organisms

• Integrative vectors: For stable, single-copy chromosomal integration of the sahase gene

• Expression enhancement elements:

  • Optimized ribosome binding sites

  • Codon-optimized gene sequences

  • Transcription terminators to prevent read-through

The pET28a+ vector has been successfully used for high-level expression of adenosylhomocysteinase from C. glutamicum in E. coli Rosetta cells, yielding more than 40 mg protein per gram of wet cells .

What are the kinetic parameters of recombinant C. glutamicum adenosylhomocysteinase?

The kinetic characterization of recombinant C. glutamicum adenosylhomocysteinase (CgrSAHase) reveals important parameters for understanding its catalytic behavior:

Table 1: Kinetic Parameters of Recombinant CgrSAHase

These Michaelis-Menten constants indicate that the enzyme has relatively high affinity for its substrates, particularly for adenosine with a K_m of only 1.4 μM. The enzyme demonstrates significant capacity for both the hydrolysis of SAH and its synthesis from adenosine and homocysteine .

The hydrolytic activity (SAH → adenosine + homocysteine) is physiologically important for maintaining methylation cycles, while the synthetic activity (adenosine + homocysteine → SAH) is of biotechnological interest for the production of SAH .

For research purposes, these kinetic parameters are essential for establishing optimal reaction conditions, designing enzyme assays, and comparing enzymatic efficiency across different experimental conditions or enzyme variants.

What purification methods yield the highest activity for recombinant C. glutamicum adenosylhomocysteinase?

The optimal purification strategy for maintaining high activity of recombinant C. glutamicum adenosylhomocysteinase involves a two-step procedure:

• Tangential ultrafiltration:

  • Initial clarification of cell lysate

  • Concentration of the protein solution

  • Removal of low molecular weight contaminants

  • Preservation of quaternary structure

• Affinity chromatography:

  • His-tag based purification using Ni-NTA or similar matrices

  • Careful optimization of imidazole concentrations:

    • Low imidazole (10-20 mM) in binding buffer to reduce non-specific binding

    • Gradient or step elution with increasing imidazole (50-300 mM)

  • Monitoring of elution fractions for both protein content and enzymatic activity

This combination has proven effective for obtaining pure, active CgrSAHase with high recovery yields. The careful execution of this two-step procedure preserves the quaternary structure of the enzyme, which is essential for its catalytic activity .

Critical factors affecting enzyme activity during purification:

• Temperature control (maintaining 4°C throughout processing)

• Buffer composition (phosphate buffer pH 7.5 with reducing agents)

• Minimizing exposure to oxidative conditions

• Addition of stabilizing agents (e.g., glycerol at 10%)

• Rapid processing to prevent proteolytic degradation

When properly purified, the enzyme exhibits a molecular weight of approximately 210 kDa by gel filtration, suggesting a tetrameric structure, while SDS-PAGE analysis reveals monomeric subunits of 52 ± 1 kDa .

How does the quaternary structure of recombinant C. glutamicum adenosylhomocysteinase compare to other bacterial SAHases?

The quaternary structure of recombinant Corynebacterium glutamicum adenosylhomocysteinase (CgrSAHase) reveals interesting structural characteristics when compared to other bacterial homologs:

• C. glutamicum SAHase structure:

  • Molecular weight by gel filtration: ~210 kDa

  • Molecular weight of monomer by SDS-PAGE: 52 ± 1 kDa

  • Predicted quaternary structure: Homotetramer (consistent with the 4:1 ratio between native and denatured molecular weights)

• Comparative analysis with other bacterial SAHases:

  Table 2: Quaternary Structure Comparison of Bacterial Adenosylhomocysteinases

  Organism	Monomer Size (kDa)	Quaternary Structure	Cofactor Requirements
  C. glutamicum	52 ± 1	Tetramer (~210 kDa)	NAD+
  E. coli	55	Tetramer	NAD+
  M. tuberculosis	54	Tetramer	NAD+
  P. aeruginosa	53	Tetramer	NAD+

  The tetrameric structure is highly conserved among bacterial SAHases, suggesting its functional importance for catalytic activity. The assembly of four subunits creates the proper active site configuration and provides stability to the enzyme under various physiological conditions.

• Structural implications:

  • The tetrameric assembly allows for cooperative substrate binding

  • The quaternary structure impacts thermal stability and pH tolerance

  • The integrity of the tetramer is crucial for maintaining catalytic efficiency

  • Subunit interfaces may present targets for structure-based enzyme engineering

Understanding these structural characteristics is essential for researchers working on protein engineering, crystallography studies, or developing inhibitors targeting this enzyme.

What promoter systems are most effective for expressing adenosylhomocysteinase in C. glutamicum?

The choice of promoter significantly impacts the expression levels and regulation of adenosylhomocysteinase in C. glutamicum. Based on research findings, several promoter systems have proven effective:

• Inducible promoters:

  • P_tac and P_lacUV5: IPTG-inducible promoters that allow tight regulation and strong expression

  • P_tet: Tetracycline-inducible system offering dose-dependent expression control

  • Advantages: Precise control over expression timing, minimizing metabolic burden during growth phase

• Auto-inducible promoters:

  • SigB-dependent cg3141 promoter system: Engineered P_4-N14 promoter activates during transition to stationary phase

  • Advantages: No need for expensive inducers, simplified scale-up in bioreactors

• Constitutive promoters:

  • P_sodA, P_tuf, P_cspB: Derived from C. glutamicum genome

  • Advantages: Continuous expression without induction requirements, varied expression strength options

Table 3: Comparison of Promoter Systems for Adenosylhomocysteinase Expression in C. glutamicum

When expressing adenosylhomocysteinase, researchers should select the promoter system based on their specific experimental needs. For detailed kinetic studies requiring high protein purity, inducible systems offer better control. For larger-scale applications, auto-inducible promoters may be more economical and practical .

What strategies can optimize the catalytic efficiency of recombinant C. glutamicum adenosylhomocysteinase?

Enhancing the catalytic efficiency of recombinant C. glutamicum adenosylhomocysteinase requires a multifaceted approach combining protein engineering, reaction condition optimization, and cofactor management:

• Protein Engineering Approaches:

  • Rational design: Based on structural analysis, modify residues in the active site or substrate binding pocket to improve substrate affinity or turnover rate

  • Directed evolution: Generate libraries of adenosylhomocysteinase variants through error-prone PCR or DNA shuffling, followed by high-throughput screening for improved catalytic properties

  • Semi-rational design: Combine structural knowledge with targeted randomization of specific residues

• Reaction Environment Optimization:

  • pH optimization: Systematically test pH ranges (typically 7.0-8.5) to identify optimal conditions for maximal activity

  • Temperature profiling: Determine temperature optimum and improve thermal stability through buffer additives or protein engineering

  • Ionic strength: Optimize salt concentration to enhance enzyme stability without compromising activity

• Cofactor Management:

  • NAD⁺ regeneration systems: Incorporate NAD⁺ recycling mechanisms to maintain cofactor availability

  • Cofactor binding optimization: Engineer the NAD⁺ binding pocket for improved affinity or reduced dissociation rate

• Expression System Refinements:

  • Codon optimization: Adjust codon usage to match tRNA abundance in C. glutamicum

  • Chaperone co-expression: Express molecular chaperones to assist proper folding

  • Post-translational modifications: Control or enhance specific modifications that improve catalytic performance

Table 4: Catalytic Optimization Strategies and Their Reported Effects

These strategies should be applied systematically, with careful evaluation of each modification's impact on the enzyme's kinetic parameters and stability profile.

How can site-directed mutagenesis improve the stability or activity of recombinant C. glutamicum adenosylhomocysteinase?

Site-directed mutagenesis offers powerful approaches to enhance the stability and activity of recombinant C. glutamicum adenosylhomocysteinase through targeted amino acid substitutions:

• Active Site Engineering:

  • Substrate binding pocket modifications: Based on the known kinetic parameters (K_m values of 12 μM for SAH, 1.4 μM for adenosine, and 40 μM for homocysteine) , mutations could target the homocysteine binding site to improve affinity

  • Catalytic residue optimization: Modify residues directly involved in the hydrolysis reaction to enhance turnover rate

  • Second-shell residue modifications: Alter amino acids that don't directly contact substrates but influence active site geometry

• Thermostability Enhancement:

  • Introduction of salt bridges: Add stabilizing ionic interactions at subunit interfaces

  • Increasing hydrophobic core packing: Replace small residues with bulkier hydrophobic amino acids in the protein core

  • Proline substitutions: Introduce prolines in loop regions to reduce conformational entropy

  • Glycine replacement: Substitute flexible glycines with more rigid amino acids to reduce unfolding entropy

• pH Tolerance Improvement:

  • Surface charge redistribution: Modify surface-exposed charged residues to optimize electrostatic interactions at target pH

  • Histidine introduction: Add histidines at strategic positions to act as pH-dependent switches

• Subunit Interface Stabilization:

  • Tetramer stabilization: Enhance intersubunit contacts to stabilize the native quaternary structure, which is essential for activity

  • Disulfide bridge introduction: Engineer carefully positioned cysteine pairs to form stabilizing disulfide bonds

Table 5: Potential Mutation Sites and Expected Outcomes

For adenosylhomocysteinase specifically, researchers should focus on maintaining the tetrameric structure (observed at ~210 kDa by gel filtration) while enhancing catalytic efficiency.

What are the challenges in achieving high-resolution structural analysis of recombinant C. glutamicum adenosylhomocysteinase?

Obtaining high-resolution structural data for recombinant C. glutamicum adenosylhomocysteinase presents several technical challenges that researchers must address:

• Protein Sample Preparation Challenges:

  • Heterogeneity: The tetrameric nature of CgrSAHase (~210 kDa) may lead to conformational heterogeneity

  • Stability during concentration: Maintaining tetrameric assembly during concentration for crystallization

  • Post-translational modifications: Potential heterogeneity from varying modification states

  • Ligand occupancy: Partial occupancy of NAD⁺ cofactor or substrate analogs

• Crystallization Obstacles:

  • Large protein size: The tetrameric structure (~210 kDa) increases crystallization difficulty

  • Dynamic regions: Flexible loops may hinder crystal formation

  • Domain movements: Conformational changes between open/closed states

  • Crystallization conditions:

    • Buffer optimization (pH 6.5-8.5)

    • Precipitant screening (PEG series, ammonium sulfate)

    • Additive testing (especially NAD⁺ and substrate analogs)

    • Temperature effects (4°C vs. room temperature)

• Diffraction Quality Issues:

  • Resolution limitations: Large unit cells may limit achievable resolution

  • Anisotropic diffraction: Uneven diffraction quality in different directions

  • Radiation damage: Sensitivity to X-ray exposure, requiring multiple crystals or special data collection strategies

• Alternative Structural Approaches:

  • Cryo-electron microscopy (cryo-EM):

    • Sample preparation (avoiding preferred orientations)

    • Image processing (dealing with conformational heterogeneity)

  • Small-angle X-ray scattering (SAXS):

    • Sample monodispersity requirements

    • Data interpretation challenges for multi-domain proteins

Table 6: Structural Analysis Methods and Their Applications to CgrSAHase

Successful structural analysis would provide invaluable insights into substrate binding, catalytic mechanism, and the basis for the enzyme's kinetic parameters (K_m values of 12 μM for SAH, 1.4 μM for adenosine, and 40 μM for homocysteine) .

How does adenosylhomocysteinase contribute to metabolic engineering applications in C. glutamicum?

Adenosylhomocysteinase plays a significant role in metabolic engineering applications of C. glutamicum, particularly in pathways involving sulfur metabolism, methylation reactions, and amino acid production:

Table 7: Metabolic Engineering Applications Involving adenosylhomocysteinase in C. glutamicum

The high expression level achieved for recombinant CgrSAHase (>40 mg protein/g wet cells) suggests that metabolic engineering applications can benefit from efficient expression of this enzyme when needed for pathway optimization in C. glutamicum.

What are the optimal conditions for measuring adenosylhomocysteinase activity in vitro?

Establishing reliable and reproducible activity assays for adenosylhomocysteinase is crucial for accurate enzyme characterization. The following conditions have been optimized for in vitro measurement of C. glutamicum adenosylhomocysteinase activity:

• Buffer System and pH:

  • Optimal buffer: 50 mM potassium phosphate buffer

  • pH range: Optimal activity at pH 7.5-8.0

  • Additional components:

    • 1 mM EDTA (chelates inhibitory metal ions)

    • 1-5 mM DTT or 2-mercaptoethanol (maintains reduced environment)

• Temperature Considerations:

  • Activity assay: Typically conducted at 30°C (physiological for C. glutamicum)

  • Thermal stability: Pre-incubation studies recommended to determine stability limits

• Substrate Concentrations:

  • For hydrolytic direction (SAH → adenosine + homocysteine):

    • SAH: 50-100 μM (>4× K_m of 12 μM)

  • For synthetic direction (adenosine + homocysteine → SAH):

    • Adenosine: 10-20 μM (>7× K_m of 1.4 μM)

    • Homocysteine: 200-400 μM (>5× K_m of 40 μM)

• Cofactor Requirements:

  • NAD⁺: 0.5-1.0 mM (ensure saturation)

  • Avoid NADH oxidation by limiting exposure to light and oxygen

• Activity Detection Methods:

  • Spectrophotometric methods:

    • Direct monitoring of adenosine at 265 nm

    • Coupled enzyme assays using adenosine deaminase

  • HPLC analysis:

    • Reversed-phase separation of substrates and products

    • Sensitive detection of SAH, adenosine, and homocysteine

• Control Reactions:

  • Enzyme-free controls to account for non-enzymatic hydrolysis

  • Heat-inactivated enzyme controls

  • Standards curves for quantification

Table 8: Troubleshooting Common Issues in Adenosylhomocysteinase Activity Assays

These optimized conditions allow for precise determination of enzyme kinetics, including the reported K_m values of 12 μM for SAH, 1.4 μM for adenosine, and 40 μM for homocysteine .

How can researchers troubleshoot low expression levels of recombinant adenosylhomocysteinase in C. glutamicum?

When facing challenges with low expression levels of recombinant adenosylhomocysteinase in C. glutamicum, researchers can implement a systematic troubleshooting approach:

• Genetic Construct Optimization:

  • Codon usage: Analyze and optimize codons for C. glutamicum preferences

  • Ribosome binding site (RBS): Modify RBS strength and spacing from start codon

  • Promoter selection: Test alternative promoters (inducible, auto-inducible, or constitutive)

  • Verify sequence integrity: Confirm absence of mutations or frameshifts

• Expression Conditions Optimization:

  • Growth medium composition:

    • Rich vs. minimal media

    • Carbon source type and concentration

    • Addition of precursor amino acids

  • Induction parameters (for inducible systems):

    • Induction timing (growth phase)

    • Inducer concentration

    • Temperature post-induction (frequently lower temperatures improve folding)

• Host Strain Considerations:

  • Protease-deficient strains: Minimize proteolytic degradation

  • Optimization of metabolic background: Select strains with appropriate metabolic flux

  • Chaperone co-expression: Add molecular chaperones to improve folding

• Process Scale-up Factors:

  • Oxygen transfer: Optimize aeration and agitation

  • pH control: Maintain optimal pH throughout cultivation

  • Feeding strategy: Implement fed-batch approaches to minimize overflow metabolism

Table 9: Systematic Approach to Troubleshooting Low Expression

For C. glutamicum specifically, researchers should consider that high-level recombinant protein expression has been demonstrated (>40 mg protein/g wet cells for CgrSAHase) , indicating that optimization can yield substantial improvements for challenging proteins.

What strategies address enzyme stability challenges during long-term storage of purified adenosylhomocysteinase?

Preserving the stability and activity of purified recombinant C. glutamicum adenosylhomocysteinase during long-term storage requires careful consideration of storage conditions and stabilizing additives:

• Storage Buffer Optimization:

  • Buffer composition:

    • 50 mM phosphate or Tris buffer, pH 7.5-8.0

    • 100-200 mM NaCl to maintain ionic strength

  • Protective additives:

    • Glycerol (20-50%): Prevents freezing damage and stabilizes protein structure

    • Reducing agents (1-5 mM DTT or 2-mercaptoethanol): Prevents oxidation of cysteine residues

    • EDTA (0.1-1 mM): Chelates metal ions that could promote oxidation

• Storage Temperature Considerations:

  • Short-term storage: 4°C (days) with preservatives

  • Medium-term storage: -20°C (months) in buffer with 25-50% glycerol

  • Long-term storage: -80°C (years) or lyophilization with cryoprotectants

• Advanced Stabilization Strategies:

  • Enzyme immobilization:

    • Attachment to solid supports increases stability

    • Methods include covalent binding, entrapment, encapsulation

  • Chemical modification:

    • Crosslinking with glutaraldehyde

    • PEGylation to reduce aggregation and increase solubility

  • Protein engineering:

    • Introduction of stabilizing mutations

    • Elimination of oxidation-prone residues

• Quality Control During Storage:

  • Regular activity testing: Monitor enzyme activity over time

  • Structural integrity assessment: Size exclusion chromatography to verify maintenance of tetrameric structure (~210 kDa)

  • Freeze-thaw minimization: Aliquot into single-use volumes before freezing

Table 10: Adenosylhomocysteinase Storage Stability Under Various Conditions

The most critical factor for maintaining the activity of adenosylhomocysteinase is preserving its tetrameric structure, as indicated by the difference between its native molecular weight (~210 kDa) and monomeric form (52 ± 1 kDa) .

How can researchers verify the correct folding and quaternary structure of recombinant adenosylhomocysteinase?

Verification of proper folding and quaternary structure is essential for ensuring the functionality of recombinant C. glutamicum adenosylhomocysteinase. The following analytical approaches provide comprehensive structural assessment:

• Size and Oligomeric State Determination:

  • Size exclusion chromatography (SEC):

    • Confirms tetrameric assembly (~210 kDa)

    • Detects aggregation or dissociation

    • Can be coupled with multi-angle light scattering (SEC-MALS) for absolute molecular weight

  • Native PAGE:

    • Preserves quaternary structure during electrophoresis

    • Comparative analysis with known standards

  • Analytical ultracentrifugation (AUC):

    • Sedimentation velocity analysis for homogeneity assessment

    • Sedimentation equilibrium for accurate molecular weight determination

• Structural Integrity Analysis:

  • Circular dichroism (CD) spectroscopy:

    • Far-UV (190-250 nm): Secondary structure content

    • Near-UV (250-350 nm): Tertiary structure fingerprint

  • Fluorescence spectroscopy:

    • Intrinsic tryptophan fluorescence for tertiary structure assessment

    • Hydrophobic dye binding (ANS, bis-ANS) for exposed hydrophobic patches

  • Differential scanning calorimetry (DSC)/fluorimetry (DSF):

    • Thermal unfolding profiles

    • Identification of stabilizing conditions

• Functional Verification:

  • Enzyme kinetics:

    • Verification of expected K_m values (12 μM for SAH, 1.4 μM for adenosine, 40 μM for homocysteine)

    • Calculation of specific activity (units/mg)

  • Cofactor binding assessment:

    • NAD⁺ binding stoichiometry

    • Spectroscopic analysis of bound cofactor

• Advanced Structural Characterization:

  • Limited proteolysis:

    • Probes accessibility of cleavage sites

    • Properly folded proteins show resistance to digestion

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Maps solvent-accessible regions

    • Identifies dynamic domains and interaction sites

Table 11: Structure Verification Methods for Adenosylhomocysteinase

The combination of these techniques provides comprehensive verification of the proper tetrameric assembly (~210 kDa) essential for adenosylhomocysteinase activity .

What are the key future research directions for recombinant C. glutamicum adenosylhomocysteinase?

The study of recombinant C. glutamicum adenosylhomocysteinase (ahcY) continues to evolve, with several promising research directions that could significantly advance our understanding and application of this enzyme:

• Structural Biology Frontiers:

  • High-resolution structural determination through X-ray crystallography or cryo-EM

  • Structure-function relationships of the tetrameric assembly (~210 kDa)

  • Conformational dynamics during catalysis

  • Molecular basis for substrate specificity (K_m values of 12 μM for SAH, 1.4 μM for adenosine, and 40 μM for homocysteine)

• Protein Engineering Opportunities:

  • Rational design for enhanced catalytic efficiency

  • Engineering substrate specificity for biotechnological applications

  • Stability enhancement for industrial applications

  • Creation of adenosylhomocysteinase variants with novel functions

• Metabolic Engineering Applications:

  • Integration into synthetic methylation pathways

  • Engineering C. glutamicum for enhanced sulfur-containing amino acid production

  • Development of adenosylhomocysteinase-based biosensors for metabolic flux analysis

  • Exploration of the enzyme's role in the broader context of C. glutamicum as an industrial amino acid producer (e.g., L-arginine production exceeding 90 g/L)

• Biotechnology Development:

  • Scale-up of recombinant adenosylhomocysteinase production

  • Immobilization strategies for biocatalysis applications

  • Continuous enzymatic processes for SAH production

  • Integration with other enzymes in multi-step biocatalytic cascades

• Fundamental Biochemistry:

  • Deeper understanding of the enzyme's role in methylation regulation

  • Exploration of potential moonlighting functions

  • Comparative analysis across bacterial phyla

  • Investigation of post-translational modifications affecting activity

The continued research into C. glutamicum adenosylhomocysteinase will likely yield valuable insights that bridge fundamental biochemical understanding with practical biotechnological applications, building upon the established biochemical characterization and the growing importance of C. glutamicum as a recombinant protein expression host .