Recombinant Opitutus terrae Serine hydroxymethyltransferase (glyA)

What is Opitutus terrae Serine hydroxymethyltransferase (glyA) and what is its primary function?

Serine hydroxymethyltransferase (SHMT) encoded by the glyA gene in Opitutus terrae is a PLP-dependent enzyme that catalyzes the reversible conversion of serine to glycine with the simultaneous conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate. This reaction is a critical component of one-carbon metabolism, providing essential one-carbon units for various biosynthetic pathways including purine and thymidylate synthesis.

The recombinant form of this enzyme from Opitutus terrae strain DSM 11246 / JCM 15787 / PB90-1 has been expressed and made available for research purposes . SHMT enzymes are highly conserved across species, but studying organism-specific variants provides insights into metabolic adaptations. In bacterial systems, SHMT plays crucial roles in amino acid metabolism and carbon utilization pathways, connecting glycine, serine, and one-carbon unit metabolism.

What is the taxonomic context of Opitutus terrae and why is it significant for SHMT research?

Opitutus terrae PB90-1 is an obligatory anaerobic bacterium belonging to the phylum Verrucomicrobia, which represents a deeply branching bacterial lineage . This phylum is interesting to researchers because its members are commonly detected in metagenomic libraries from diverse environments including aquatic, terrestrial, and intestinal ecosystems, yet they are rarely cultured in laboratory settings .

Opitutus terrae was isolated specifically from rice paddy soil and is capable of producing propionate from plant-derived polysaccharides . This metabolic capability makes its enzymes, including SHMT, particularly interesting for studying adaptations to anaerobic soil environments. The genome of Opitutus terrae PB90-1 has been fully sequenced, revealing a circular chromosome of 5,957,605 bp with a G+C content of 65.3% .

Studying SHMT from this organism offers insights into:

• Metabolic adaptations in anaerobic soil bacteria

• Evolution of one-carbon metabolism in the Verrucomicrobia phylum

• Functional diversity of SHMT across bacterial lineages

How does bacterial SHMT contribute to amino acid metabolism pathways?

SHMT plays a crucial role in bacterial amino acid metabolism, particularly in the interconversion of serine and glycine. Studies with SHMT from other bacteria, such as Corynebacterium glutamicum, demonstrate that this enzyme can also catalyze the aldol cleavage of threonine to glycine, although at a lower rate than its primary reaction with serine .

In C. glutamicum, SHMT demonstrated an aldol cleavage activity with L-threonine at 1.3 μmol min⁻¹ (mg of protein)⁻¹, which was approximately 4% of its activity with L-serine as substrate . This secondary activity has significant implications for amino acid metabolism and production, as it represents a potential degradation pathway for threonine.

The role of SHMT in these metabolic networks makes it a target of interest for metabolic engineering studies. For example, researchers working with C. glutamicum were able to increase L-threonine production by 49% by reducing SHMT activity to 8% of its initial levels, which correspondingly reduced glycine production by 41% . Similar metabolic relationships likely exist in Opitutus terrae, making its SHMT an interesting target for metabolic studies.

What are the recommended handling procedures for recombinant Opitutus terrae SHMT?

For optimal results when working with recombinant Opitutus terrae SHMT, researchers should follow these handling procedures :

Storage conditions:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot for long-term storage at -20°C/-80°C

Shelf life considerations:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

Proper handling is critical for maintaining enzyme activity and ensuring reproducible experimental results.

What are the optimal assay conditions for measuring Opitutus terrae SHMT enzymatic activity?

Based on studies with SHMT enzymes from related organisms, researchers can design appropriate assay conditions for Opitutus terrae SHMT. While specific conditions for O. terrae SHMT are not directly provided in the search results, the following methodology can be adapted from work with other bacterial SHMTs:

Standard SHMT activity assay:

• Buffer system: 50 mM potassium phosphate buffer, pH 7.5

• Temperature: 30-37°C (optimize based on the organism's growth temperature)

• Required cofactors: Pyridoxal 5'-phosphate (PLP) at 50-100 μM

• Substrates: L-serine (1-10 mM) and tetrahydrofolate (0.1-1 mM)

Spectrophotometric assay approach:
The reaction can be monitored by coupling the formation of 5,10-methylenetetrahydrofolate to the reduction of methenyltetrahydrofolate by NADPH, which can be measured spectrophotometrically at 340 nm.

Alternative assay for aldol cleavage activity:
For measuring the secondary activity (threonine degradation), researchers can use L-threonine as substrate and measure glycine formation using specific analytical methods such as HPLC or enzymatic coupled assays .

How can researchers investigate the substrate specificity of Opitutus terrae SHMT?

Investigating substrate specificity is crucial for understanding the functional characteristics of Opitutus terrae SHMT. Based on research with other bacterial SHMTs, the following methodological approach is recommended:

Comparative enzyme kinetics methodology:

• Prepare purified recombinant O. terrae SHMT using affinity chromatography

• Conduct enzyme assays with various potential substrates:

  • L-serine (primary substrate)

  • L-threonine (potential secondary substrate)

  • Other structurally similar amino acids

Analysis of kinetic parameters:
For each substrate, determine:

• Km (substrate affinity)

• kcat (catalytic rate constant)

• kcat/Km (catalytic efficiency)

Example data table format for substrate specificity:

From studies with C. glutamicum SHMT, we know that its aldol cleavage activity with L-threonine was approximately 4% of its activity with L-serine . Similar comparative studies with O. terrae SHMT would provide valuable insights into its substrate preferences and potential metabolic roles.

What strategies can be employed for optimizing expression and purification of Opitutus terrae SHMT?

For researchers seeking to produce their own recombinant Opitutus terrae SHMT, the following optimization strategies are recommended:

Expression system optimization:

• Host selection: While the commercial protein is expressed in mammalian cells , bacterial expression systems (E. coli) may be more accessible for academic research

• Vector design: Include appropriate affinity tags (His-tag is commonly used) and optimize codon usage for the chosen expression host

• Induction conditions: Test various temperatures (16-37°C), inducer concentrations, and induction times

Purification protocol:

• Affinity chromatography: Using appropriate tag-based purification (e.g., Ni-NTA for His-tagged proteins)

• Secondary purification: Consider ion exchange or size exclusion chromatography for higher purity

• Buffer optimization: Test various buffer compositions to maintain enzyme stability (including PLP as cofactor)

Quality control:

• Verify purity using SDS-PAGE (aim for >85% purity as with commercial preparations)

• Confirm protein identity using mass spectrometry

• Validate enzyme activity using standard SHMT assays

Researchers can modify the tag type based on their specific experimental needs, as noted in the commercial protein specifications .

How can site-directed mutagenesis be utilized to study structure-function relationships in Opitutus terrae SHMT?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in enzymes like Opitutus terrae SHMT. Based on general SHMT knowledge and approaches used with other bacterial enzymes, researchers can employ the following methodology:

Target selection for mutagenesis:

• Active site residues: Identify and mutate key catalytic residues based on sequence alignments with well-characterized SHMTs

• Substrate binding pocket: Modify residues involved in substrate recognition to alter specificity

• PLP binding site: Investigate residues involved in cofactor binding

• Oligomerization interfaces: Examine the role of protein-protein interactions in enzyme function

Mutagenesis protocol outline:

• Design primers for PCR-based site-directed mutagenesis

• Perform mutagenesis using standard molecular biology techniques

• Express and purify mutant proteins using the same protocol as wild-type

• Characterize mutants through:

  • Structural analysis (circular dichroism, thermal stability)

  • Enzyme kinetics (Km, kcat, substrate specificity)

  • Oligomerization state (size exclusion chromatography)

Potential mutagenesis targets based on SHMT conservation:

• PLP-binding lysine residue

• Catalytic residues involved in proton abstraction

• Substrate recognition residues in the active site pocket

This approach would provide mechanistic insights into how Opitutus terrae SHMT functions and potentially reveal unique features compared to other bacterial SHMTs.

What are the applications of isotope labeling studies with Opitutus terrae SHMT in metabolic research?

Isotope labeling studies provide powerful tools for investigating the role of SHMT in metabolic pathways. For Opitutus terrae SHMT, researchers can apply the following methodological approaches:

In vitro isotope tracing methodologies:

• ¹³C-labeled serine experiments: Trace the transfer of labeled carbons to glycine and one-carbon units

• ²H-labeled substrates: Investigate hydrogen exchange during catalysis

• ¹⁵N-labeled amino acids: Examine nitrogen metabolism connections

Data analysis approaches:

• Mass spectrometry for detecting labeled metabolites

• NMR spectroscopy for structural confirmation

• Flux analysis to quantify metabolic rates

Application in metabolic context:
Since Opitutus terrae is capable of fermenting plant-derived polysaccharides , isotope labeling could reveal connections between carbon utilization pathways and one-carbon metabolism mediated by SHMT.

Example experiment design:

• Incubate purified O. terrae SHMT with ¹³C-labeled serine and tetrahydrofolate

• Analyze products using LC-MS/MS to detect labeled glycine and 5,10-methylenetetrahydrofolate

• Quantify isotopomer distributions to determine reaction mechanisms

These methodologies allow researchers to understand the precise role of SHMT in the metabolic network of Opitutus terrae and potentially uncover novel metabolic pathways.

How does the anaerobic lifestyle of Opitutus terrae influence the function and regulation of its SHMT?

Opitutus terrae is an obligatory anaerobic bacterium isolated from rice paddy soil , and this anaerobic lifestyle likely has significant implications for the function and regulation of its SHMT enzyme.

Metabolic adaptations in anaerobic environments:

• In anaerobic organisms, SHMT may be integrated with different metabolic pathways compared to aerobic bacteria

• The enzyme may have evolved specific regulatory mechanisms tied to redox state or alternative electron acceptors

• The connection between one-carbon metabolism and anaerobic respiration or fermentation pathways represents an interesting research area

Research methodology for investigating anaerobic adaptations:

• Comparative biochemistry: Compare kinetic properties of O. terrae SHMT with those from aerobic bacteria

• Redox sensitivity analysis: Examine enzyme activity under various redox conditions

• Metabolic context studies: Investigate how SHMT activity connects to propionate production from plant polysaccharides, a known metabolic capability of O. terrae

Potential research questions:

• Does O. terrae SHMT show altered substrate specificity compared to aerobic counterparts?

• Are there anaerobic-specific post-translational modifications or regulatory mechanisms?

• How is SHMT activity coordinated with other metabolic pathways under anaerobic conditions?

Understanding these adaptations could provide insights into specialized metabolic strategies employed by anaerobic soil bacteria.

What approaches can be used to study the role of Opitutus terrae SHMT in its ecological context?

Understanding the role of SHMT in the ecological context of Opitutus terrae requires integrating biochemical knowledge with ecological and genomic approaches:

Genomic context analysis:

• Examine the genomic neighborhood of the glyA gene in O. terrae to identify potentially co-regulated genes

• Compare with related organisms to identify conserved gene clusters

• The complete genome sequence of O. terrae PB90-1 (GenBank accession number CP001032) provides the basis for these analyses

Experimental ecological approaches:

• Microcosm studies: Examine gene expression of glyA under various soil conditions

• Co-culture experiments: Investigate interactions with methanogens, which are known to interact with Opitutus spp. in response to hydrogen partial pressures

• Metabolomic profiling: Measure metabolites related to one-carbon metabolism in environmental samples

Functional genomics methodologies:

• RNA-seq to measure glyA expression under various conditions

• Proteomics to quantify SHMT protein levels in different growth states

• Metabolic modeling to predict the role of SHMT in the organism's adaptation to rice paddy soil

These integrated approaches would help researchers understand how SHMT contributes to the ecological fitness of Opitutus terrae in its natural environment and potentially reveal novel metabolic interactions.