Recombinant Chloroflexus aurantiacus Serine hydroxymethyltransferase (glyA)

What is the basic function of Chloroflexus aurantiacus Serine hydroxymethyltransferase (glyA)?

Chloroflexus aurantiacus Serine hydroxymethyltransferase (glyA) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate (THF) serving as the one-carbon carrier. This reaction represents a major source of one-carbon groups required for the biosynthesis of purines, thymidylate, methionine, and other important biomolecules. Additionally, the enzyme exhibits THF-independent aldolase activity toward beta-hydroxyamino acids, producing glycine and aldehydes through a retro-aldol mechanism . This dual functionality makes glyA a versatile enzyme in C. aurantiacus metabolism, contributing to both amino acid interconversion and one-carbon transfer reactions essential for cellular function.

What are the structural characteristics of C. aurantiacus glyA protein?

The C. aurantiacus glyA protein belongs to the SHMT (Serine hydroxymethyltransferase) family and consists of 419 amino acids with a molecular mass of approximately 45.1 kDa . The complete amino acid sequence is:

MLEHLRATDPIIADLIEREAQRQRQGLELIASENYTSLAVMEAQGSVLTNKYAEGLPGRRYYGGCEFVDAIEQLAIERACQLFGTSHANVQPHSGAQANIAVFTALLQPGDTILGMRLDHGGHLTHGSPVNFSGKWYNVHFYGVDAQTGQIDYDDLASKARAIRPKLITSGASAYPRIIDFARMRQIADEVGALLMADIAHIAGLVAAGEHPSPVGHAHVITTTTHKTLRGPRGGLILMGDDFAKQLNSSVFPGTQGGPLMHVIAGKAVAFGEALRPEFRQYAAQIRRNARALAEGLMAQGLTLVSGGTDNHLMLVDLRSTGLTGAQAQRALDKAAITVNKNAIPDDPQPPMKTSGIRIGTPAVTTRGMREPEMAQIAAWIGEVLMYPDDEARLNRIAGEVADLCRHFPVPADMVQVRG

While detailed crystal structure information isn't provided in the search results, the protein likely adopts the typical fold of SHMT family members, with conserved catalytic residues and binding sites for pyridoxal 5'-phosphate (PLP), the essential cofactor for SHMT activity.

How does C. aurantiacus glyA contribute to the organism's unique metabolism?

As a thermophilic filamentous anoxygenic phototrophic bacterium, C. aurantiacus possesses unique metabolic capabilities, including the ability to grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions . The glyA enzyme plays a critical role in this metabolic versatility by supporting:

• One-carbon metabolism essential for nucleotide synthesis

• Amino acid interconversion between serine and glycine

• Potential involvement in carbon fixation pathways, notably the 3-HP bi-cycle that has been identified in C. aurantiacus and successfully integrated into E. coli

The ability of C. aurantiacus to thrive in diverse environmental conditions depends partly on these fundamental metabolic processes supported by glyA. Additionally, as Chloroflexi species represent the earliest branching bacteria capable of photosynthesis according to 16S rRNA analysis, studying their core metabolic enzymes like glyA provides insights into the evolution of photosynthetic and carbon-fixing metabolic pathways .

What are the kinetic parameters of recombinant C. aurantiacus glyA compared to orthologs from other organisms?

Researchers working with recombinant C. aurantiacus glyA should consider comparative kinetic analysis with orthologs from mesophilic and other thermophilic organisms. While specific kinetic parameters aren't provided in the search results, typical parameters to investigate include:

Given C. aurantiacus' thermophilic nature, its glyA likely exhibits enhanced thermostability and potentially different catalytic parameters compared to mesophilic counterparts. The thermostability properties make this enzyme particularly valuable for biotechnological applications requiring high-temperature reactions.

How can recombinant C. aurantiacus glyA be integrated into artificial carbon fixation pathways?

The integration of recombinant C. aurantiacus glyA into artificial carbon fixation pathways represents an advanced research area with significant implications for synthetic biology. The 3-HP bi-cycle from C. aurantiacus has already been successfully integrated into E. coli , suggesting that key enzymes from this organism can function in heterologous hosts.

For researchers exploring this application, several considerations are important:

• The role of glyA in one-carbon metabolism makes it potentially valuable in pathways that involve glycine-dependent carbon fixation, such as the reductive glycine (rGly) pathway.

• Integration strategies should consider:

  • Optimization of expression conditions considering the thermophilic origin

  • Potential need for chaperones to ensure proper folding in mesophilic hosts

  • Metabolic balancing to ensure sufficient THF regeneration

  • Coupling with formate assimilation pathways for complete carbon fixation

• Specific research approaches might include:

  • Co-expression with other C. aurantiacus enzymes to reconstruct partial or complete metabolic modules

  • Directed evolution to enhance activity at lower temperatures for mesophilic hosts

  • Protein engineering to optimize catalytic efficiency for specific synthetic pathways

The ability of C. aurantiacus glyA to function in synthetic carbon fixation systems could contribute to the development of microorganisms capable of using C1 compounds (CO2, formate, methanol) as sole carbon sources, with applications in biofuel production and carbon capture technologies .

What are the effects of temperature and pH on the catalytic efficiency and stability of recombinant C. aurantiacus glyA?

As C. aurantiacus is a thermophilic organism, its glyA enzyme likely exhibits unique temperature-dependent properties that require careful characterization. Researchers should investigate:

• Temperature-activity profile:

  • Expected higher temperature optimum compared to mesophilic SHMTs

  • Potential activity across a broader temperature range

  • Relationship between temperature and reaction specificity (SHMT vs. aldolase activity)

• Thermostability characteristics:

  • Half-life at different temperatures

  • Mechanisms of thermostability (e.g., increased hydrophobic interactions, salt bridges)

  • Protein unfolding kinetics using techniques like differential scanning calorimetry

• pH-dependent properties:

  • Optimal pH for both forward and reverse reactions

  • pH-stability profile

  • Changes in substrate specificity with pH

These parameters are crucial for optimizing expression and purification protocols, as well as for designing experimental conditions for enzyme characterization and application development.

What expression systems are optimal for producing functional recombinant C. aurantiacus glyA?

The choice of expression system for recombinant C. aurantiacus glyA requires careful consideration of several factors:

• E. coli expression systems:

  • The most common approach for initial expression attempts

  • Recommended strains: BL21(DE3), Rosetta, or Arctic Express (for cold expression)

  • Vector considerations: pET series vectors with T7 promoter for high expression

  • Induction strategies: IPTG concentration optimization (0.1-1.0 mM) and temperature adjustment (often lower temperatures of 16-25°C improve folding of thermophilic proteins)

  • Co-expression with chaperones may be necessary, as demonstrated for other thermophilic proteins like Rubisco from T. denitrificans, which required E. coli chaperones GroEL and GroES for proper folding

• Alternative expression hosts:

  • Thermophilic hosts like Thermus thermophilus may provide better folding environments

  • Yeast systems (S. cerevisiae, P. pastoris) if post-translational modifications are needed

  • Cell-free expression systems for rapid screening of expression conditions

• Fusion tags and purification strategies:

  • Affinity tags (His6, GST, MBP) for simplified purification

  • Cleavable tags if native protein is required for activity assays

  • Heat treatment (e.g., 60-70°C) as an initial purification step, taking advantage of the thermostable nature of the enzyme

• Activity verification:

  • Spectrophotometric assays tracking the formation of 5,10-methylene-THF

  • Coupled enzyme assays with 5,10-methylene-THF-dependent enzymes

  • Isotope exchange assays for kinetic measurements

Each expression approach should be evaluated based on yield, solubility, purity, and specific activity of the recombinant enzyme.

How can the dual activities of C. aurantiacus glyA (SHMT and THF-independent aldolase) be measured and distinguished experimentally?

Differentiating between the SHMT and THF-independent aldolase activities of C. aurantiacus glyA requires careful experimental design:

• SHMT activity measurement:

  • Spectrophotometric assays monitoring the formation of 5,10-methylene-THF (absorption at 240 nm)

  • Coupled enzyme assays where 5,10-methylene-THF is used by a downstream enzyme

  • Isotope labeling with 14C or 13C to track carbon transfer

  • HPLC or LC-MS methods to detect and quantify serine-glycine interconversion

• THF-independent aldolase activity measurement:

  • Direct detection of aldehyde formation using chemical trapping agents

  • HPLC analysis of reaction products

  • Spectrophotometric assays coupled with aldehyde dehydrogenase

  • Mass spectrometry to identify products from various β-hydroxyamino acid substrates

• Differentiation strategies:

  • Perform assays in the presence and absence of THF

  • Use of specific inhibitors for each activity

  • Substrate specificity analysis using various β-hydroxyamino acids

  • Site-directed mutagenesis of residues predicted to affect one activity more than the other

• Data analysis for dual activity:

These methodologies allow researchers to characterize the bifunctional nature of the enzyme and investigate potential regulatory mechanisms between the two activities.

What approaches can be used to investigate the role of C. aurantiacus glyA in metabolic engineering applications?

Investigating C. aurantiacus glyA in metabolic engineering requires systematic approaches:

• Heterologous expression strategies:

  • Integration into model organisms (E. coli, S. cerevisiae)

  • Expression optimization using different promoters and RBS strengths

  • Codon optimization for host organisms

  • Balancing expression with other pathway enzymes

• Pathway integration methods:

  • Construction of synthetic operons containing glyA and related genes

  • Pathway modularization approaches

  • Integration with carbon fixation pathways like the 3-HP bicycle or rGly pathway

  • Genomic integration vs. plasmid-based expression

• Performance evaluation techniques:

  • Metabolic flux analysis using 13C labeling

  • Growth phenotype characterization

  • Product yield determination

  • Adaptive laboratory evolution (ALE) to optimize pathway performance, as demonstrated for other carbon fixation pathways

• Analytical methods for pathway analysis:

  • LC-MS/MS for metabolite profiling

  • Transcriptomics and proteomics to assess system-wide effects

  • Isotope tracing to follow carbon flow through engineered pathways

  • Real-time monitoring of pathway activity using biosensors

Researchers should consider that the thermostability of C. aurantiacus glyA might provide advantages in metabolic engineering applications, potentially allowing for process conditions at elevated temperatures or providing greater enzyme longevity in continuous processes.

What are the main challenges in expressing and characterizing recombinant C. aurantiacus glyA?

Several challenges exist in working with recombinant C. aurantiacus glyA:

• Expression challenges:

  • Thermophilic proteins often fold poorly in mesophilic hosts

  • Potential toxicity if the enzyme disturbs host amino acid metabolism

  • Codon usage bias between C. aurantiacus and expression hosts

  • Need for specific cofactors (PLP) during expression and purification

• Characterization difficulties:

  • Distinguishing between the dual catalytic activities

  • Limited availability of comparative data from related thermophilic SHMTs

  • Potential requirement for specialized equipment for high-temperature assays

  • Stability of reagents (especially THF) at elevated temperatures

• Application development issues:

  • Balancing thermostability with catalytic activity at desired operating temperatures

  • Integration with other enzymes that may have different temperature optima

  • Potential substrate channeling requirements when incorporated into metabolic pathways

  • Scale-up considerations for biotechnological applications

Addressing these challenges requires interdisciplinary approaches combining protein engineering, biophysical characterization, and metabolic modeling.

How might protein engineering improve the utility of C. aurantiacus glyA for biotechnological applications?

Protein engineering offers several avenues to enhance C. aurantiacus glyA for biotechnological applications:

• Stability engineering:

  • Further enhancement of thermostability for industrial processes

  • Improving stability in organic solvents or at extreme pH

  • Engineering for compatibility with immobilization techniques

• Catalytic property modifications:

  • Altering substrate specificity to accept non-natural amino acids

  • Enhancing the efficiency of either SHMT or aldolase activity

  • Modifying cofactor requirements or improving cofactor binding

• Compatibility improvements:

  • Engineering variants that perform optimally at lower temperatures for mesophilic hosts

  • Reducing product inhibition for continuous processes

  • Creating fusion proteins for substrate channeling in synthetic pathways

• Directed evolution strategies:

  • Error-prone PCR to generate diversity

  • Screening for activity under desired conditions

  • Selection systems coupling enzyme activity to cell survival

  • Semi-rational approaches targeting specific regions based on structural information

These engineering efforts could significantly expand the utility of C. aurantiacus glyA in applications ranging from biocatalysis to synthetic carbon fixation pathways.