Recombinant Saccharomyces cerevisiae CTP synthase 2 (URA8), partial

What is the relationship between URA7 and URA8 genes in Saccharomyces cerevisiae?

The URA7 and URA8 genes encode CTP synthase enzymes in S. cerevisiae that share 70% identity at the nucleotide level, while their deduced amino acid sequences (Ura7p and Ura8p) show 78% identity . Neither gene is individually essential provided that cells possess at least one functional CTP synthase-encoding gene. The URA7 mRNA is approximately 2-fold more abundant than the URA8 transcript . In a ura8 mutant, CTP levels are only 22% lower than wild type, whereas in a ura7 mutant, CTP levels drop by 64%, indicating that the URA7-encoded enzyme is responsible for the majority of CTP produced in vivo .

How do the structural characteristics of URA8 compare to those of URA7?

Both URA7 and URA8-encoded CTP synthases have been purified to homogeneity and characterized regarding their kinetic and enzymological properties . The purified enzymes exist as dimers in solution and oligomerize to form tetramers in the presence of their substrates UTP and ATP . The subunit molecular masses are very similar: 64.7 kDa for Ura7p and 64.5 kDa for Ura8p . Recent cryo-EM studies have revealed that both enzymes can form filaments, with Ura8 filaments yielding higher resolution structures (2.8 Å for substrate-bound and 3.8 Å for product-bound states) compared to Ura7 filaments .

What kinetic differences exist between URA7 and URA8 enzymes?

Despite their sequence similarity, the two enzymes exhibit different kinetic properties. The URA7-encoded enzyme shows positive cooperative kinetics when the UTP concentration is varied at a saturating concentration of ATP or when the ATP concentration is varied at a saturating concentration of UTP . In contrast, the URA8-encoded CTP synthase exhibits positive cooperative kinetics only when the UTP concentration is varied at subsaturating concentrations of ATP or when the ATP concentration is varied at subsaturating concentrations of UTP .

At physiological concentrations of UTP (0.75 mM) and ATP (2.3 mM), which are considered saturating for both enzymes, URA8 would be expected to exhibit saturation kinetics toward these substrates, while URA7 would continue to show positive cooperativity . This suggests that the two enzymes may be differentially regulated in vivo through fluctuations in substrate concentrations.

How can researchers express and purify recombinant URA8 for structural and functional studies?

For the expression and purification of recombinant URA8, researchers have successfully used multicopy plasmid systems in S. cerevisiae . The methodology typically includes:

• Constructing expression plasmids containing the URA8 gene with a C-terminal His6-tag, similar to the approach used for human CTP synthase expression in yeast .

• Transforming an appropriate S. cerevisiae strain with the expression plasmid. For complementation studies, researchers can use strain SDO195 (an ura7Δ ura8Δ double mutant bearing a URA7 plasmid) and select for transformants that can grow after losing the URA7 plasmid through 5-fluoroorotic acid (5FOA) selection .

• Growing the transformed yeast in appropriate media to induce expression of the recombinant protein.

• Lysing cells and purifying the His-tagged URA8 protein using nickel affinity chromatography, followed by additional purification steps as needed.

This approach allows for the isolation of functional enzyme suitable for kinetic, structural, and regulatory studies.

What experimental approaches enable the study of URA8 filament formation and structure?

Researchers have employed cryo-electron microscopy (cryo-EM) to determine the structures of URA8 filaments with high resolution. The methodology includes:

• Assembling filaments at pH 6.5, where single filaments predominate over larger bundles, facilitating structure determination .

• Preparing filaments in both substrate-bound (ATP and UTP) and product-bound (CTP) states for comparative structural analysis .

• Using cryo-EM data collection and processing to solve structures at resolutions of 2.8 Å (substrates) and 3.8 Å (products) for URA8 filaments .

• For comparison, determining the structure of free URA8 tetramers at pH 7.4 bound to substrates at 2.8 Å resolution .

These approaches have revealed important insights into the conformational changes associated with enzyme regulation and filament formation.

How can researchers investigate the role of specific residues in URA8 function through mutagenesis?

Site-directed mutagenesis studies have identified key residues that are conserved across CTP synthases from various species including E. coli, C. trachomatis, B. subtilis, and S. cerevisiae . For URA8, researchers can:

• Target conserved residues such as Glu 161 and His 233 (equivalent positions in both URA7 and URA8) through site-directed mutagenesis.

• Introduce specific mutations, such as E161K, which has been shown to significantly affect CTP synthase activity and reduce sensitivity to product inhibition .

• Express the mutant proteins in appropriate yeast strains (including complementation of ura7Δ ura8Δ double mutants) to assess functional consequences in vivo.

• Purify the mutant proteins for in vitro characterization of kinetic properties, tetramerization capacity, and response to regulatory molecules.

This approach allows for structure-function analysis and identification of residues critical for catalysis, regulation, or filament formation.

How is URA8 activity regulated through post-translational modifications?

While most phosphorylation studies have focused on URA7, the mechanisms may apply to URA8 as well given their sequence similarity. Based on the literature:

• CTP synthase is phosphorylated on multiple serine residues in vivo .

• Protein kinases A and C have been shown to phosphorylate CTP synthase, resulting in stimulation of enzyme activity through increased catalytic turnover .

• Phosphorylation facilitates the nucleotide-dependent tetramerization of the enzyme and decreases its sensitivity to feedback inhibition by CTP .

• When human CTP synthase (CTPS1) is expressed in S. cerevisiae, it is also phosphorylated by both protein kinase A and protein kinase C, suggesting conservation of this regulatory mechanism .

Researchers investigating URA8 regulation should consider analyzing its phosphorylation state under various conditions and assessing how these modifications affect enzyme activity and filament formation.

What is the significance of the conformational differences observed between substrate-bound and product-bound URA8 filaments?

Cryo-EM structures have revealed important differences between substrate-bound and product-bound URA8 filaments:

• The primary difference is the conformation of individual CTPS protomers. Active and inactive conformations are characterized by a ~7° rotation of the glutaminase domain relative to the amido-ligase domain .

• This rotation opens an ammonia channel between the two active sites in a single protomer in the active conformation .

• The URA8 product-bound filament adopts the canonical inhibited conformation, while the substrate-bound filament structure exists in an intermediate state between canonical active and inhibited conformations, with the ammonia channel remaining closed .

• In contrast, free URA8 tetramers (not in filaments) bound to substrates at pH 7.4 adopt the canonical active conformation .

These findings suggest that one function of yeast CTPS filaments is to constrain the enzyme in a low activity conformation, providing an additional regulatory mechanism beyond allosteric control.

How does product inhibition regulate URA8 activity?

CTP synthase enzymes are allosterically regulated through product inhibition by CTP . For researchers investigating this mechanism:

• CTP binding causes conformational changes that reduce enzyme activity.

• Mutations that alleviate this regulation (such as E161K) result in elevated cellular levels of CTP and increased phospholipid synthesis via the Kennedy pathway .

• The sensitivity to CTP inhibition can be modulated by phosphorylation, with phosphorylated enzyme showing decreased sensitivity to feedback inhibition .

• In filament structures, the product-bound state adopts an inhibited conformation, suggesting that filament formation may enhance product inhibition .

Understanding this regulatory mechanism is particularly important for studies involving metabolic engineering or for investigating conditions that might alter cellular CTP levels.

What experimental conditions should be optimized when comparing URA7 and URA8 activities?

When designing experiments to compare URA7 and URA8 activities, researchers should consider:

• Substrate concentrations: Given the different kinetic properties, experiments should include both saturating and subsaturating concentrations of UTP and ATP to capture the distinct behaviors of the two enzymes .

• pH conditions: pH can affect both enzyme activity and filament formation. For instance, URA8 filaments form preferentially at pH 6.5, while higher pH may favor the active tetrameric form .

• Phosphorylation state: The activity of both enzymes is regulated by phosphorylation, so the presence of phosphatases or kinases (or their inhibitors) may significantly impact measured activities .

• Oligomerization state: Since the tetrameric form is the active state, conditions that influence tetramerization will affect activity measurements .

• Product inhibition: The presence of CTP can inhibit enzyme activity, and the two enzymes may differ in their sensitivity to this inhibition .

Table 1: Comparison of URA7 and URA8 properties

How can researchers accurately assess URA8 filament formation in vivo and in vitro?

For in vivo assessment:

• Fluorescent tagging: URA8 can be tagged with fluorescent proteins to visualize its distribution and potential filament formation in living cells using fluorescence microscopy.

• Environmental conditions: Researchers should investigate how factors such as pH, nutrient availability, and cellular stress affect filament formation.

For in vitro assessment:

• Electron microscopy: Negative staining EM or cryo-EM can be used to directly visualize filament formation under different conditions .

• Light scattering: Changes in light scattering can be used to monitor the assembly of URA8 into larger structures.

• pH dependence: Assembly at pH 6.5 has been shown to favor single filaments rather than bundles, which is advantageous for structural studies .

Table 2: Structural data for URA8 filaments and tetramers

What approaches can resolve discrepancies in URA8 activity measurements between different experimental setups?

When faced with contradictory results in URA8 activity measurements, researchers should consider:

How can URA8 be used as a model system for studying human CTP synthase function?

The functional expression of human CTP synthase genes (CTPS1 and CTPS2) in S. cerevisiae provides a valuable experimental system:

• Complementation assays: Human CTPS1 and CTPS2 genes have been shown to complement the lethal phenotype of the ura7Δ ura8Δ double mutant, indicating functional conservation .

• Post-translational regulation: Human CTP synthase expressed in yeast is phosphorylated in a manner similar to the native yeast enzyme, suggesting conservation of regulatory mechanisms .

• Structural insights: The relatively high degree of sequence identity (~53%) between yeast and human enzymes makes yeast an excellent model for studying structural aspects of CTP synthase function .

• Drug screening: Yeast expressing human CTP synthase can be used to screen for compounds that affect enzyme activity, potentially identifying new therapeutic approaches for conditions where CTP synthase is dysregulated.

This system provides researchers with a genetically tractable model for investigating fundamental aspects of CTP synthase function that may be relevant to human health and disease.