Recombinant Synechococcus sp. Ribose-phosphate pyrophosphokinase (prs)

What is ribose-phosphate pyrophosphokinase and what is its metabolic significance in Synechococcus sp.?

Ribose-phosphate pyrophosphokinase (encoded by the prs gene) catalyzes the transfer of the β,γ-diphosphate from ATP to the C1 hydroxyl group of ribose-5-phosphate (R5P) to form 5-phosphoribosyl-1-pyrophosphate (PRPP). In Synechococcus sp., PRPP serves as a critical metabolic intermediate connecting several essential biosynthetic pathways:

• Nucleotide biosynthesis: PRPP is required for both de novo and salvage pathways of purine and pyrimidine nucleotides

• Amino acid biosynthesis: PRPP is utilized in the synthesis of histidine and tryptophan

• NAD biosynthesis: PRPP is essential for the biosynthesis of pyridine nucleotide coenzymes

• Carbon metabolism integration: Links photosynthetic carbon fixation to nucleic acid synthesis

In photosynthetic organisms like Synechococcus, PRPP synthase plays a particularly important role as R5P can be derived from the Calvin cycle, creating a direct connection between carbon fixation and nucleotide metabolism .

How do researchers typically express recombinant Synechococcus prs in laboratory settings?

Expression of recombinant Synechococcus prs typically employs the following methodological approaches:

E. coli expression systems:

• Most commonly used due to ease of genetic manipulation and high protein yields

• Often employs pET vectors with T7 promoter systems for controlled expression

• Can be verified through complementation of E. coli Δprs mutations

• May require optimization to address potential inclusion body formation

Expression optimization strategies:

• Lower temperatures (15-25°C) to improve protein folding

• Reduced inducer concentrations

• Co-expression with chaperones like GroEL/GroES

• Use of solubility-enhancing fusion tags (MBP, SUMO)

Purification approaches:

• If inclusion bodies form, solubilization with urea (6-8M) followed by gradual removal to restore activity

• Inclusion of proper cofactors (Mg²⁺, ATP) during refolding

• Combination of ion exchange (e.g., DEAE-cellulose) and size exclusion chromatography

Based on similar cyanobacterial protein expression patterns, researchers should monitor for inclusion body formation and optimize accordingly, as was observed with Synechococcus phytoene desaturase, where the recombinant protein comprised 5% of total cellular protein but was predominantly found in the inclusion body fraction .

What assay methods are recommended for measuring Synechococcus prs activity?

Several complementary methods can be employed to measure recombinant Synechococcus prs activity:

Direct PRPP formation assays:

• Coupling PRPP formation with nucleotide synthesis using purified enzymes

• HPLC analysis of PRPP formation using appropriate separation conditions

• LC-MS methods for precise quantification of reaction products

Phosphate release assays:

• Colorimetric detection of pyrophosphate release using the Malachite Green assay

• This approach has been successfully used for other Synechococcus phosphate-utilizing enzymes

Coupled enzyme assays:

• Linking PRPP synthesis to NADH oxidation via coupling enzymes

• Monitoring absorbance decrease at 340 nm due to NADH consumption

Standard assay conditions typically include:

• Buffer system (typically HEPES or Tris) at pH 7.5-8.0

• Divalent metal ions (Mg²⁺ is common; Mn²⁺ may enhance activity in some cases)

• Ribose-5-phosphate and ATP as substrates

• Inorganic phosphate (especially for phosphate-dependent variants)

• Reducing agent (DTT or β-mercaptoethanol)

When designing assays, consider that PRPP synthases from different sources may show differential dependence on inorganic phosphate. As noted in research with spinach PRPP synthases, some isoforms required inorganic phosphate for activity while others were phosphate-independent .

How does the structure of Synechococcus prs compare to PRPP synthases from other organisms?

While specific structural data for Synechococcus prs is limited, comparative analysis with characterized PRPP synthases reveals:

Domain organization:

• Conservation of catalytic domain for binding ribose-5-phosphate, ATP, and divalent cations

• Preservation of "PRPP binding site" and "flexible loop" motifs found in other PRPP synthases

• Potential unique regulatory domains adapted to photosynthetic metabolism

Quaternary structure:

• Most bacterial PRPP synthases function as hexamers

• Synechococcus prs likely adopts a similar oligomeric structure

• Interfaces between subunits may contain regulatory sites, as seen in human PRS1 where an allosteric site was identified at the dimer interface

Metal ion coordination:

• Coordination of divalent cations (Mg²⁺ or Mn²⁺) is crucial for activity

• In human PRS1, a metal ion binds at the active site and interacts with ATP phosphates

• The metal binding preferences may differ between organisms, potentially reflecting environmental adaptations

Phosphate binding:

• Variable dependency on inorganic phosphate among PRPP synthases

• Some spinach PRPP synthase isoforms required inorganic phosphate while others were phosphate-independent

• This variability may represent adaptation to fluctuating environmental phosphate levels

For structural characterization, approaches should include X-ray crystallography, site-directed mutagenesis of predicted key residues, and comparative molecular dynamics simulations with other bacterial PRPP synthases.

What is known about the regulation of Synechococcus prs activity in vivo?

Regulation of Synechococcus prs activity involves multiple mechanisms coordinating enzyme function with cellular metabolism:

Allosteric regulation:

• PRPP synthases are typically regulated by ADP (product inhibition) and inorganic phosphate (activation)

• In photosynthetic organisms, additional regulators may include Calvin cycle intermediates

• Glyceraldehyde-3-phosphate (GAP) has been shown to activate certain Calvin cycle enzymes in Synechococcus and may similarly affect prs

Environmental influences:

• Phosphate availability significantly impacts nucleotide metabolism in cyanobacteria

• Under phosphate-deplete conditions, cyanophages show altered infection dynamics, suggesting broad metabolic reprogramming in the host

• If Synechococcus prs is phosphate-dependent, its activity would be directly reduced during phosphate limitation

Light and redox regulation:

• As photosynthetic organisms, Synechococcus metabolism is strongly influenced by light

• Redox state fluctuates with photosynthetic activity and may regulate enzyme function

• Addition of reducing agents (DTT) and oxidizing agents (DTNB) affects proteins in Synechococcus, including several metabolic enzymes

Metabolic coordination:

• Integration with carbon fixation via the Calvin cycle

• Coordination with nucleotide biosynthesis pathways

• Balance with amino acid synthesis (particularly histidine and tryptophan)

To study these regulatory mechanisms, researchers should employ approaches including site-directed mutagenesis of predicted allosteric sites, enzyme kinetics studies with various metabolites, and thermal shift assays to detect ligand binding.

How does phosphate limitation affect prs expression and activity in Synechococcus sp.?

Phosphate limitation has significant impacts on prs function in Synechococcus, reflecting the central role of phosphate in PRPP synthesis:

Direct effects on enzyme activity:

• Some PRPP synthase isoforms require inorganic phosphate for activity

• If Synechococcus prs is phosphate-dependent, its activity would be directly reduced during limitation

• This creates a regulatory mechanism linking nucleotide synthesis to phosphate availability

Transcriptional responses:

• Under phosphate-deplete conditions, cyanophage infection dynamics are altered, suggesting broad metabolic reprogramming

• Phosphate limitation typically induces expression of the Pho regulon, which may indirectly affect prs transcription

Adaptation mechanisms:

• Cyanophages infecting Synechococcus have acquired host genes encoding phosphate-binding proteins (PstS)

• This adaptation is thought to improve virus replication under phosphate starvation

• Similar adaptations may exist for maintaining PRPP synthesis during limitation

Methodological approaches:

• Growth experiments under controlled phosphate concentrations

• Direct enzyme activity measurements from cells grown at different phosphate levels

• Transcriptomic analysis to measure prs expression changes

• Metabolomic analysis to track changes in PRPP and related metabolites

The importance of phosphate in Synechococcus metabolism is highlighted by the finding that cyanophage S-PM2d infection of Synechococcus sp. WH7803 during phosphate-deplete conditions showed evidence of perturbed infection dynamics, notably an extended latent period .

How can genetic engineering of the prs gene improve metabolic productivity in Synechococcus?

Strategic engineering of the prs gene offers several approaches to enhance metabolic productivity:

Overexpression strategies:

• Constitutive or inducible overexpression to increase PRPP availability

• Coordinated expression with downstream pathways that utilize PRPP

• In Synechococcus elongatus PCC 7942, a combined approach of metabolite doping and metabolic engineering successfully improved production of 2-phenylethanol

Regulatory modifications:

• Engineering prs variants resistant to feedback inhibition

• This approach is similar to what was done with aroG and pheA genes in engineered Synechococcus, where feedback inhibition resistant (fbr) versions significantly improved production

• Mutation of allosteric sites to create constitutively active variants

Balancing with downstream pathways:

• Co-expression of prs with downstream enzymes that utilize PRPP

• Avoiding metabolic bottlenecks by ensuring balanced pathway flux

• For example, in S. elongatus PCC 7942, researchers combined overexpression of shikimate kinase with targeted metabolite supplementation to overcome bottlenecks

Optimizing for cultivation conditions:

• Engineering prs variants with properties suited to specific cultivation conditions

• Creating variants less sensitive to phosphate limitation

• Adaptation to light regimes used in photobioreactors

A successful precedent for this approach is seen in the recombinant S. elongatus PCC 7942 p120 strain, where researchers implemented phenylalanine-doped growth medium to overcome metabolic load while simultaneously engineering the strain to increase carbon flow into the shikimate pathway .

What role does the prs gene play in the essential gene set of Synechococcus?

The prs gene is a critical component of the essential gene repertoire in Synechococcus:

Evidence for essentiality:

• In Synechococcus elongatus PCC 7942, comprehensive transposon mutagenesis identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions

• As a key enzyme in nucleotide biosynthesis with no alternative pathways, prs likely belongs to this essential gene set

• The absence of viable prs knockout mutants supports this classification

Integration with core metabolism:

• In photosynthetic organisms, prs links carbon fixation to nucleotide metabolism

• This creates a direct connection between photosynthesis and DNA/RNA synthesis

• The essentiality of this linkage is heightened in obligate photoautotrophs

Evolutionary conservation:

• Essential genes typically show tight overlap with well-conserved genes across species

• PRPP synthase is highly conserved across diverse organisms

• The conservation pattern supports its fundamental role in cellular metabolism

Contextual essentiality:

• While prs is likely unconditionally essential, its importance may be heightened under certain conditions

• During rapid growth or nucleotide stress, prs function becomes even more critical

• The interaction between prs and conditionally essential genes varies with environmental conditions

For studying gene essentiality in Synechococcus, researchers have successfully employed high-density transposon mutagenesis libraries. For example, in S. elongatus PCC 7942, a library of ~250,000 transposon mutants was created and sequenced to identify insertion locations, enabling the identification of essential genes based on the absence of viable insertions .

How do Calvin cycle intermediates interact with PRPP synthase in Synechococcus?

The interaction between Calvin cycle intermediates and PRPP synthase represents a critical regulatory node connecting photosynthesis with nucleotide metabolism:

Potential regulatory interactions:

• Calvin cycle intermediates may serve as allosteric regulators of PRPP synthase

• In Synechococcus, glyceraldehyde-3-phosphate (GAP) has been shown to activate certain Calvin cycle enzymes, specifically fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase)

• Similar interactions may occur with prs to coordinate nucleotide synthesis with photosynthetic activity

Experimental evidence from related enzymes:

• Limited proteolysis small molecule mapping (LiP-SMap) has detected hundreds of metabolite-protein interactions in cyanobacterial proteomes

• The Calvin cycle intermediate GAP activated both Synechocystis and Cupriavidus F/SBPase by reducing KM, while the GAP isomer dihydroxyacetone phosphate (DHAP) had no effect

• This substrate specificity suggests similar precise regulatory interactions may exist with PRPP synthase

Methodological approaches:

• In vitro enzyme assays with various Calvin cycle metabolites

• Thermal shift assays to detect binding-induced conformational changes

• LiP-SMap techniques to identify metabolite-protein interactions on a proteome-wide scale

• Site-directed mutagenesis of predicted binding sites

The LiP-SMap technique has successfully identified metabolite-protein interactions in the proteomes of two cyanobacteria (Synechocystis sp. PCC 6803 and Synechococcus PCC 7942) and revealed that some metabolites interact in a species-specific manner .

What are the challenges in purifying active recombinant Synechococcus prs?

Purification of active recombinant Synechococcus prs presents several technical challenges:

Inclusion body formation:

• Cyanobacterial proteins expressed in E. coli often form inclusion bodies

• Similar to Synechococcus phytoene desaturase, which was predominantly found in the inclusion body fraction (constituting 5% of total cellular protein)

• This necessitates optimized solubilization and refolding strategies

Refolding protocols:

• For Synechococcus phytoene desaturase, urea was successfully used to solubilize the protein from inclusion bodies

• The protein was subsequently purified on a DEAE-cellulose column with 40% recovery of the original protein

• Enzyme activity was restored upon removal of urea, suggesting similar approaches may work for prs

Potential toxicity:

• Expression of active enzymes involved in nucleotide metabolism can disrupt host cell physiology

• Similar challenges were observed with cyanophage genome fragments, where plasmids containing certain fragments could not be transformed into E. coli, suggesting toxicity

• Tightly controlled inducible promoters or expression of inactive mutants may address this issue

Cofactor requirements:

• PRPP synthases have specific requirements for divalent metal ions

• For some enzymes, the choice of metal ion significantly impacts activity

• While most PRPP synthases prefer Mg²⁺, other enzymes like the Lreu_1276 protein exhibited maximum activity with Mn²⁺

Oligomeric structure preservation:

• PRPP synthases typically function as oligomers

• Purification conditions must preserve quaternary structure

• Size-exclusion chromatography can be used to verify proper oligomerization

What are the kinetic differences between phosphate-dependent and phosphate-independent PRPP synthase variants?

PRPP synthases from different sources display varying phosphate dependencies with distinct kinetic properties:

Phosphate dependency patterns:

• Some PRPP synthases require inorganic phosphate for activity while others are phosphate-independent

• In spinach, two PRPP synthase isozymes (1 and 2) required inorganic phosphate for activity, whereas two others (3 and 4) were phosphate-independent

• These differences likely reflect evolutionary adaptations to different cellular environments

Kinetic parameters:

Structural basis:

• Differences in phosphate binding sites between variants

• Potential alternate mechanisms for stabilizing reaction intermediates

• Synechococcus PRPP synthase isozymes may possess distinct structural features related to their phosphate dependency

Experimental assessment:

• Activity assays with and without phosphate supplementation

• Kinetic characterization across a range of phosphate concentrations

• Mutagenesis of predicted phosphate binding residues

In experiments with spinach PRPP synthases expressed in E. coli, clear differences were observed: isozymes 1 and 2 showed high activity with phosphate but minimal activity without it, while isozymes 3 and 4 maintained activity regardless of phosphate presence .

How does the expression of recombinant Synechococcus prs compare in heterologous versus homologous systems?

The choice between heterologous and homologous expression systems presents important trade-offs for recombinant Synechococcus prs:

Heterologous expression in E. coli:

In E. coli, expression challenges can be significant. For Synechococcus phytoene desaturase, the recombinant protein was predominantly found in inclusion bodies, requiring solubilization with urea . Similar issues may arise with prs.

Homologous expression in Synechococcus:

Optimization strategies:

• For E. coli: Expression at lower temperatures, co-expression with chaperones, use of solubility tags

• For Synechococcus: Use of strong light-inducible or constitutive promoters, optimization of culture conditions

Purification considerations:

• From E. coli: May require refolding protocols if inclusion bodies form

• From Synechococcus: Additional steps may be needed to remove photosynthetic pigments

The choice should be guided by the research goals: E. coli expression is preferable for structural studies requiring large protein amounts, while homologous expression is better for functional studies in a native-like environment.