Recombinant Shewanella woodyi Phosphoserine aminotransferase (serC)

What is phosphoserine aminotransferase (serC) and what is its function in cellular metabolism?

Phosphoserine aminotransferase (PSAT, encoded by the serC gene) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate-linked reversible transamination reaction. This reaction proceeds through a bimolecular ping-pong mechanism and is a critical part of the phosphorylated pathway of serine biosynthesis . In this pathway, serC functions as the second enzyme in a three-step process where 3-phosphoglycerate (3-PGA) derived from glycolysis or the Calvin cycle is converted to serine. The biological significance of this enzyme lies in its essential role in amino acid metabolism, particularly serine biosynthesis, which is crucial for protein synthesis and various cellular processes.

What experimental approaches can be used to confirm the enzymatic activity of recombinant Shewanella woodyi serC?

Several experimental approaches can verify the enzymatic activity of recombinant S. woodyi serC:

• Complementation assays: Expressing S. woodyi serC in E. coli serC mutants to restore growth in serine-dropout medium, similar to experiments performed with M. tuberculosis serC .

• Spectrophotometric assays: Monitoring the transamination reaction through:

  • Coupled enzyme assays with glutamate dehydrogenase to measure α-ketoglutarate production

  • Direct measurement of PLP to PMP conversion by tracking absorbance changes at 330-420 nm

• HPLC or LC-MS/MS analysis: Direct quantification of 3-phosphoserine production from 3-phosphohydroxypyruvate and glutamate.

• Isotope labeling studies: Using isotope-labeled substrates to track the transfer of amino groups during the transamination reaction.

These approaches collectively provide robust confirmation of enzymatic activity and mechanistic insights.

What are the optimal expression systems and conditions for producing soluble, active Shewanella woodyi serC?

Based on available information about recombinant S. woodyi proteins, several expression systems can be used for producing serC :

• E. coli expression system:

  • Recommended strains: BL21(DE3), Rosetta, or Arctic Express for challenging proteins

  • Growth temperature: Lower temperatures (16-20°C) post-induction often improve solubility

  • Induction conditions: 0.1-0.5 mM IPTG, OD600 of 0.6-0.8

  • Media supplementation: Add 50-100 μM PLP to ensure cofactor incorporation

  • Expression vectors: pET series with appropriate fusion tags (His6, GST, MBP)

• Alternative expression systems include yeast, baculovirus, and mammalian cell systems, as noted in the product description .

For PLP-dependent enzymes like serC, maintaining cofactor availability throughout expression and purification is crucial for obtaining active enzyme. Optimizing these conditions through small-scale expression trials before scaling up production is highly recommended.

What purification strategy yields the highest recovery of active Shewanella woodyi serC?

An effective purification strategy for recombinant S. woodyi serC should include:

• Initial capture step:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged serC

  • Buffers containing 50-100 μM PLP, 1-5 mM β-mercaptoethanol or DTT, and 10% glycerol

• Intermediate purification:

  • Ion exchange chromatography based on the theoretical pI of S. woodyi serC

  • Buffer optimization to maintain enzyme stability

• Polishing step:

  • Size exclusion chromatography to isolate the properly folded dimeric form

  • Analysis by SDS-PAGE to confirm ≥85% purity as specified for commercial preparations

Throughout purification, monitoring enzymatic activity is essential to track recovery of active enzyme. The purification protocol should aim to maintain the native dimeric state of the enzyme, which is critical for its catalytic function.

How can researchers troubleshoot common expression and purification challenges with Shewanella woodyi serC?

When encountering challenges with S. woodyi serC expression and purification, consider these troubleshooting approaches:

• Low expression yield:

  • Optimize codon usage for the expression host

  • Test multiple promoter strengths and induction conditions

  • Consider fusion partners that enhance solubility (MBP, SUMO, TrxA)

  • Evaluate co-expression with molecular chaperones

• Protein insolubility:

  • Lower induction temperature (16°C overnight)

  • Reduce inducer concentration

  • Test different lysis buffers with various salt concentrations and additives

  • Consider refolding strategies if inclusion bodies persist

• Loss of enzymatic activity:

  • Ensure PLP supplementation in all buffers

  • Minimize exposure to oxidizing conditions

  • Include stabilizing agents (glycerol, trehalose)

  • Avoid excessive dilution of the enzyme

• Aggregation during purification:

  • Include mild detergents (0.01-0.05% Triton X-100)

  • Optimize buffer pH and ionic strength

  • Consider adding arginine or proline as aggregation suppressors

Systematic approach to these challenges can significantly improve the yield and quality of purified recombinant S. woodyi serC.

What are the recommended methods for determining the kinetic parameters of Shewanella woodyi serC?

Determining kinetic parameters of S. woodyi serC requires careful experimental design:

• Steady-state kinetic analysis:

  • Measure initial velocities across varying concentrations of both substrates (3-PHP and glutamate)

  • Plot data using appropriate enzyme kinetic models (ping-pong bi-bi mechanism)

  • Determine Km, kcat, and catalytic efficiency (kcat/Km) values using non-linear regression

• Pre-steady-state kinetics using stopped-flow spectroscopy:

  • Monitor rapid changes in absorbance upon mixing enzyme with substrates

  • Characterize individual steps in the catalytic cycle

  • Identify rate-limiting steps in the reaction mechanism

• Temperature and pH dependence studies:

  • Measure enzyme activity across temperature ranges (particularly important for psychrophilic Shewanella woodyi)

  • Determine pH optimum and pH-rate profiles

  • Analyze activation energy using Arrhenius plots

• Inhibition studies:

  • Evaluate product inhibition patterns

  • Test substrate analogs as potential inhibitors

  • Determine inhibition constants and mechanisms

These approaches provide comprehensive kinetic characterization essential for understanding the catalytic mechanism and comparing with serC enzymes from other organisms.

How can researchers analyze the catalytic mechanism of Shewanella woodyi serC using spectroscopic techniques?

Spectroscopic techniques provide valuable insights into the catalytic mechanism of S. woodyi serC:

• UV-visible spectroscopy:

  • Monitor PLP-enzyme absorbance (~420 nm for internal aldimine)

  • Track conversion to PMP form (~330 nm) during catalysis

  • Observe formation and decay of reaction intermediates

• Stopped-flow spectroscopy:

  • Capture transient intermediates in the catalytic cycle

  • Determine rates of individual steps in the reaction

  • Correlate spectral changes with specific catalytic events

• Circular dichroism (CD):

  • Analyze secondary structure content and stability

  • Monitor protein conformational changes during catalysis

  • Assess thermal stability and unfolding transitions

• Fluorescence spectroscopy:

  • Utilize intrinsic tryptophan fluorescence to monitor conformational changes

  • Track PLP fluorescence changes during catalysis

  • Employ FRET techniques with labeled substrates or enzyme variants

These spectroscopic approaches, when combined with site-directed mutagenesis, provide a detailed understanding of the reaction mechanism at the molecular level.

What structural analysis techniques provide the most valuable insights into Shewanella woodyi serC function?

Multiple structural analysis techniques can elucidate S. woodyi serC function:

As demonstrated with Arabidopsis thaliana PSAT1, capturing multiple catalytic states (internal aldimine, PMP form, and geminal diamine intermediate) provides comprehensive insights into the reaction mechanism .

How can site-directed mutagenesis be used to investigate critical residues in Shewanella woodyi serC catalysis?

Site-directed mutagenesis is a powerful approach for investigating S. woodyi serC catalysis:

• Target residue selection based on:

  • Sequence alignment with well-characterized PSATs

  • Structural homology modeling

  • Evolutionary conservation analysis

  • Predicted catalytic and substrate-binding residues

• Strategic mutation types:

  • Conservative substitutions (e.g., K→R, D→E) to probe specific functional groups

  • Alanine substitutions to remove side chain functionality

  • Introduction of non-canonical amino acids for precise functional testing

• Comprehensive mutant characterization:

  • Kinetic parameters (Km, kcat, kcat/Km)

  • Spectroscopic properties of enzyme-PLP complexes

  • Thermal stability and pH dependence

  • Crystal structures of key mutants

• Specific residues to target include:

  • The catalytic lysine that forms a Schiff base with PLP

  • Residues that interact with the phosphate group of PLP

  • Residues involved in substrate binding

  • Residues that stabilize the dimeric interface

This approach has been successfully applied to other phosphoserine aminotransferases and can reveal the molecular basis of catalysis in S. woodyi serC.

What strategies can be employed to crystallize Shewanella woodyi serC for structural studies?

Crystallizing S. woodyi serC for structural studies requires specialized approaches:

• Protein preparation strategies:

  • Ensure high protein homogeneity through rigorous purification

  • Maintain constant PLP saturation

  • Consider limited proteolysis to remove flexible regions

  • Verify proper oligomeric state through SEC-MALS

• Crystallization optimization:

  • Screen diverse crystallization conditions using automated systems

  • Test both vapor diffusion and microbatch methods

  • Explore crystallization at lower temperatures (4-15°C) given S. woodyi's psychrophilic nature

  • Add substrates or substrate analogs for co-crystallization

• Capturing catalytic intermediates:

  • React crystals with substrates through soaking experiments

  • Use substrate analogs or inhibitors to trap specific states

  • Employ microspectrophotometry to verify intermediate formation in crystals

• Enhancing crystal quality:

  • Employ seeding techniques to improve crystal size and diffraction

  • Test cryoprotectant conditions carefully to minimize damage

  • Consider crystal dehydration to improve diffraction quality

These approaches have proven successful for related enzymes, including PSAT from Arabidopsis thaliana, which yielded structures in multiple catalytic states .

How might Shewanella woodyi serC be engineered for enhanced catalytic properties or novel substrate specificity?

Engineering S. woodyi serC for enhanced properties can employ several strategies:

• Rational design approaches:

  • Structure-guided mutations to improve substrate binding

  • Engineering the active site architecture for novel substrate acceptance

  • Enhancing protein stability through computational design

  • Modifying residues that influence the electrostatic environment of the active site

• Directed evolution methods:

  • Error-prone PCR to generate diverse variants

  • DNA shuffling with serC genes from related organisms

  • Site-saturation mutagenesis of active site residues

  • High-throughput screening or selection systems for desired properties

• Semi-rational design combining:

  • Computational prediction of beneficial mutations

  • Focused library generation around key residues

  • Consensus approaches based on sequence alignments

• Potential engineering goals:

  • Cold adaptation enhancement for biotechnological applications

  • Broadened substrate specificity for non-canonical amino acid synthesis

  • Increased thermostability for industrial applications

  • Altered cofactor specificity or reduced cofactor dependence

These engineering approaches could yield specialized variants of S. woodyi serC for diverse research and biotechnological applications.

How does the serine biosynthesis pathway in Shewanella woodyi compare to pathways in other bacterial species?

The serine biosynthesis pathway in Shewanella woodyi likely involves three key enzymes similar to other bacteria:

• 3-phosphoglycerate dehydrogenase (PGDH): Catalyzes the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate using NAD+ as a cofactor.

• Phosphoserine aminotransferase (PSAT, serC): Converts 3-phosphohydroxypyruvate to 3-phosphoserine through transamination with glutamate .

• Phosphoserine phosphatase (PSP): Dephosphorylates 3-phosphoserine to yield serine.

Comparative analysis with other bacterial systems reveals:

• Regulatory differences: In Mycobacterium tuberculosis, CRP(Mt) directly activates serC expression by binding to the serC-Rv0885 intergenic region .

• Metabolic integration: The pathway connects primary carbon metabolism (glycolysis) with amino acid biosynthesis.

• Functional conservation: The M. tuberculosis serC can complement E. coli serC mutants, demonstrating functional conservation across bacterial species .

• Environmental adaptation: In psychrophilic bacteria like Shewanella woodyi, enzymes in this pathway may exhibit cold adaptation features.

Understanding these comparative aspects provides insights into metabolic adaptation across diverse bacterial species.

What evidence exists for the role of serC in bacterial adaptation to specific environmental niches?

Several lines of evidence suggest serC plays important roles in bacterial adaptation:

• Growth under nutrient limitation:

  • In M. tuberculosis, serC overexpression or serine supplementation accelerates growth of crp mutants in mycomedium .

  • This suggests serC is crucial for growth under specific nutritional conditions.

• Temperature adaptation:

  • As a psychrophilic marine bacterium, S. woodyi likely has adapted its metabolic enzymes, including serC, to function optimally at lower temperatures.

  • Enzymes from psychrophilic organisms often show higher catalytic efficiency at low temperatures compared to mesophilic counterparts.

• Stress response:

  • Serine biosynthesis can be upregulated under certain stress conditions, suggesting serC may play a role in stress adaptation.

  • The phosphorylated pathway provides metabolic flexibility when other amino acid synthesis routes are compromised.

• Metabolic versatility:

  • Shewanella species are known for their metabolic versatility, particularly regarding electron acceptors.

  • The serine biosynthesis pathway connects to central carbon metabolism, potentially contributing to this metabolic flexibility.

Further investigation into S. woodyi serC regulation and kinetic properties would provide deeper insights into its role in environmental adaptation.

Data Table: Comparing Phosphoserine Aminotransferases Across Species

*Estimated values based on related enzymes. Specific data for S. woodyi serC would require experimental determination.

This table highlights the conserved features across phosphoserine aminotransferases while noting potential species-specific adaptations. The dimeric structure and PLP dependency are highly conserved, while kinetic parameters and temperature optima vary across different ecological niches.

What are the most promising approaches for studying the regulatory mechanisms controlling serC expression in Shewanella woodyi?

Several complementary approaches could elucidate the regulatory mechanisms controlling serC expression in S. woodyi:

• Transcriptomic analysis:

  • RNA-seq under various growth conditions (temperature, nutrient availability, oxygen levels)

  • Identification of co-regulated genes and potential operonic structure

  • Temporal expression patterns during growth phases

• Promoter analysis:

  • 5' RACE to define transcription start sites

  • Reporter gene fusions to identify regulatory elements

  • Electrophoretic mobility shift assays to identify transcription factor binding

  • DNase footprinting to precisely map protein-DNA interactions

• Chromatin immunoprecipitation (ChIP-seq):

  • Identification of transcription factors that bind the serC promoter region

  • Genome-wide mapping of regulatory interactions

  • Integration with transcriptomic data to build regulatory networks

• Computational approaches:

  • Comparative genomics to identify conserved regulatory elements

  • Prediction of transcription factor binding sites

  • Integration of multiple omics datasets to infer regulatory networks

These approaches would reveal whether S. woodyi serC is regulated similarly to M. tuberculosis (where CRP(Mt) directly activates serC expression) or through distinct mechanisms adapted to its unique ecological niche.

How might studying Shewanella woodyi serC contribute to understanding cold adaptation mechanisms in enzymes?

Investigating S. woodyi serC as a model for cold adaptation could reveal important insights:

• Structural adaptations:

  • Reduced number of proline residues and increased glycine content for flexibility

  • Fewer ion pairs and hydrogen bonds for reduced rigidity

  • Modified surface charge distribution for improved solvent interactions at low temperatures

  • Altered active site architecture balancing flexibility and catalytic efficiency

• Kinetic adaptations:

  • Lower activation energy compared to mesophilic homologs

  • Higher kcat at low temperatures

  • Modified substrate binding affinity across temperature ranges

  • Characteristic temperature-activity profiles and thermal stability curves

• Molecular dynamics studies:

  • Simulation of enzyme flexibility at different temperatures

  • Identification of regions with enhanced mobility

  • Analysis of water coordination and hydrophobic interactions

• Comparative analysis:

  • Systematic comparison with serC from mesophilic and thermophilic organisms

  • Identification of convergent evolutionary strategies for cold adaptation

  • Site-directed mutagenesis to test hypothesized cold-adaptive features

These studies would contribute to the broader understanding of enzymatic cold adaptation and could inform enzyme engineering for low-temperature biotechnological applications.

What are the key considerations for researchers beginning work with Recombinant Shewanella woodyi Phosphoserine aminotransferase?

Researchers beginning work with recombinant S. woodyi serC should consider several key factors:

• Expression and purification strategy:

  • Select appropriate expression systems based on research needs

  • Ensure PLP supplementation throughout purification

  • Verify enzyme activity and proper folding

  • Maintain the native dimeric state of the enzyme

• Experimental design:

  • Standard assay conditions must be established and validated

  • Consider S. woodyi's psychrophilic nature when designing temperature conditions

  • Include proper controls for enzymatic assays

  • Account for cofactor dependency in all experiments

• Comparative context:

  • Benchmark against well-characterized phosphoserine aminotransferases

  • Consider evolutionary context and potential cold adaptations

  • Interpret results in light of S. woodyi's unique ecological niche

• Technical challenges:

  • PLP-dependent enzymes require special handling to maintain cofactor binding

  • Psychrophilic enzymes may show reduced stability at room temperature

  • Standardize storage conditions to maintain consistent enzyme activity

These considerations will help researchers establish robust experimental systems and generate reliable, reproducible data when working with this enzyme.

What are the broader implications of studying Shewanella woodyi serC for understanding metabolic adaptation and enzyme evolution?

Studying S. woodyi serC offers insights into several broader scientific questions:

• Metabolic adaptation to extreme environments:

  • Understanding how essential metabolic pathways adapt to cold marine environments

  • Elucidating the balance between enzyme stability and catalytic efficiency

  • Revealing evolutionary strategies for maintaining metabolic function under suboptimal conditions

• Enzyme evolution across diverse ecological niches:

  • Tracing the evolutionary history of phosphoserine aminotransferases

  • Identifying conserved catalytic mechanisms versus adaptive variations

  • Understanding the constraints and flexibility in enzyme evolution

• Implications for biotechnology:

  • Development of cold-active enzymes for industrial applications

  • Insights for protein engineering to enhance catalytic properties

  • Potential applications in bioremediation leveraging Shewanella's unique metabolic capabilities

• Fundamental enzymology:

  • Deeper understanding of PLP-dependent transamination mechanisms

  • Insights into structure-function relationships in aminotransferases

  • Contributions to protein dynamics and catalysis models