Recombinant Prochlorococcus marinus GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is GMP synthase (guaA) and what is its functional significance in Prochlorococcus marinus?

GMP synthase [glutamine-hydrolyzing] (guaA, EC 6.3.5.2) is a critical enzyme in purine nucleotide biosynthesis. In Prochlorococcus marinus, it catalyzes the ATP-dependent conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP) using glutamine as a nitrogen donor. The enzyme contains two functional domains: a glutamine amidotransferase domain and a pyrophosphatase domain, which form a single polypeptide chain in bacteria including Prochlorococcus marinus .

This enzyme is particularly significant in P. marinus because this cyanobacterium has evolved to thrive in nutrient-limited oceanic environments with a streamlined genome. The efficient functioning of nucleotide biosynthesis pathways, including GMP synthesis, is crucial for the organism's survival in these challenging conditions .

What are the recommended storage and reconstitution protocols for the recombinant protein?

The recombinant Prochlorococcus marinus GMP synthase should be stored at -20°C, with extended storage recommended at -20°C or -80°C. Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For working aliquots, storage at 4°C for up to one week is suitable .

For reconstitution:

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

• Reconstitute 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 default) for long-term storage

• Aliquot to minimize freeze-thaw cycles

The shelf life for liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms maintain stability for up to 12 months at these temperatures .

What are the structural features of the partial recombinant Prochlorococcus marinus GMP synthase?

The partial recombinant Prochlorococcus marinus GMP synthase (strain MIT 9301) covers the expression region 1-528 and has a purity of >85% as determined by SDS-PAGE . Its amino acid sequence begins with "MSQKSFKKER DPSILILDFG SQYSELIARR IRETNVFSLV VSNCISIEDI..." and includes key functional regions responsible for its catalytic activity .

The protein contains the characteristic domains found in bacterial GMP synthases: a glutamine amidotransferase domain that hydrolyzes glutamine to generate ammonia, and a synthetase domain that transfers the ammonia to XMP to form GMP. In Prochlorococcus marinus, as in other bacteria, these domains exist on a single polypeptide chain, unlike in some eukaryotes where they may be separate proteins .

What experimental approaches can be used to assess GMP synthase activity in vitro?

Table 1: Recommended experimental conditions for GMP synthase activity assays

For optimal results, researchers should begin with these baseline conditions and optimize parameters for the specific recombinant Prochlorococcus marinus GMP synthase preparation. Activity can be monitored by measuring either the consumption of substrates (XMP, glutamine) or the formation of products (GMP, glutamate) .

How does the expression system affect the functionality of the recombinant protein?

The recombinant Prochlorococcus marinus GMP synthase described in the technical information is expressed in a mammalian cell system , which differs from the native cyanobacterial expression environment. This heterologous expression can affect several aspects of protein functionality:

• Post-translational modifications: Mammalian expression systems may introduce modifications not present in the native cyanobacterial protein.

• Folding dynamics: The folding environment in mammalian cells differs from that in cyanobacteria, potentially affecting the tertiary structure.

• Protein solubility: The mammalian expression system may yield different solubility characteristics compared to the native protein.

• Enzymatic activity: While the catalytic mechanism remains the same, the specific activity may differ from the native enzyme.

Researchers should consider these factors when interpreting experimental results and, if necessary, compare the recombinant protein's properties with those of the native enzyme to identify any significant differences in functionality .

How is GMP synthase activity related to nitrogen metabolism in Prochlorococcus marinus?

Prochlorococcus marinus has evolved in nutrient-limited oceanic environments and displays specialized adaptations in its nitrogen metabolism. Unlike many cyanobacteria, P. marinus SS120 lacks genes for transport systems for nitrate, nitrite, cyanate, and urea, as well as the corresponding nitrate/nitrite reductases and urease. Instead, it relies on reduced nitrogen compounds such as NH₄⁺ and amino acids for growth .

GMP synthase plays a critical role in this specialized nitrogen economy by:

• Efficiently utilizing glutamine as a nitrogen donor for purine synthesis

• Contributing to the organism's adaptation to low-nitrogen environments

• Integrating nitrogen metabolism with nucleotide biosynthesis

The glutamine amidotransferase domain of GMP synthase is particularly significant in this context, as it directly interfaces with amino acid metabolism by utilizing glutamine. The evolutionary retention of this enzyme in P. marinus' streamlined genome underscores its essential role in the organism's survival strategy in oligotrophic oceanic environments .

How does GMP synthase function differ among Prochlorococcus ecotypes adapted to different ocean depths?

Table 2: Comparative analysis of GMP synthase characteristics across Prochlorococcus ecotypes

Prochlorococcus marinus ecotypes have adapted to different ocean depths, with corresponding variations in light intensity, temperature, and nutrient availability. Deep-adapted ecotypes like SS120 have genomic adaptations for surviving in low-light, relatively nutrient-rich deeper waters, while surface ecotypes are adapted to high-light, nutrient-poor surface waters .

What are the structural and functional implications of the glutamine amidotransferase and pyrophosphatase domains being encoded on a single polypeptide chain?

In Prochlorococcus marinus, as in other bacteria, the glutamine amidotransferase and pyrophosphatase domains of GMP synthase are encoded on a single polypeptide chain . This arrangement has several significant structural and functional implications:

• Substrate channeling: The physical proximity of the two domains facilitates the transfer of ammonia from the glutamine amidotransferase domain to the synthetase domain, protecting this reactive intermediate from the cellular environment.

• Coordinated regulation: The single polypeptide structure ensures stoichiometric production of both domains and allows for coordinated allosteric regulation.

• Evolutionary optimization: The fusion of these domains likely represents an evolutionary optimization for efficient nucleotide biosynthesis, particularly important in organisms with streamlined genomes like Prochlorococcus marinus.

• Interdomain communication: The single polypeptide structure enables direct communication between domains, potentially allowing for activity in one domain to influence the other through conformational changes.

These structural features make bacterial GMP synthases, including that of P. marinus, excellent targets for studying interdomain communication and substrate channeling in multi-domain enzymes .

What methodological approaches are appropriate for studying the impact of environmental stressors on GMP synthase function?

Table 3: Methodological approaches for studying environmental stress effects on GMP synthase

For studying how environmental factors affect GMP synthase function in Prochlorococcus marinus, researchers should consider both in vitro approaches using the purified recombinant enzyme and in vivo approaches using whole-cell cultures. The integration of these approaches can provide insights into how this essential enzyme adapts to the varying conditions experienced by different Prochlorococcus ecotypes across ocean depths and geographical regions .

What challenges and strategies exist for structural studies of Prochlorococcus marinus GMP synthase?

Structural studies of Prochlorococcus marinus GMP synthase face several challenges:

• Protein stability: As a multi-domain enzyme from a marine organism, maintaining stability during purification and crystallization can be difficult.

• Domain flexibility: The interdomain regions may exhibit flexibility that complicates crystallization.

• Expression and purification: Obtaining sufficient quantities of properly folded, active protein for structural studies can be challenging.

Strategies to address these challenges include:

• Construct optimization: Creating truncated constructs or individual domains that may crystallize more readily.

• Co-crystallization: Attempting crystallization with substrates, products, or inhibitors to stabilize specific conformations.

• Alternative structural techniques: Employing cryo-electron microscopy, small-angle X-ray scattering, or nuclear magnetic resonance for proteins resistant to crystallization.

• Computational approaches: Using homology modeling based on related GMP synthases with known structures to predict structural features.

• Stabilizing mutations: Introducing mutations that enhance protein stability without affecting function.

These approaches can help overcome the inherent challenges in studying the structure of this important enzyme from an ecologically significant marine cyanobacterium .

How does the amino acid sequence and structure of Prochlorococcus marinus GMP synthase compare to homologs in other organisms?

The Prochlorococcus marinus GMP synthase (guaA) amino acid sequence "MSQKSFKKER DPSILILDFG SQYSELIARR..." reveals conservation of key functional residues typical of bacterial GMP synthases . When comparing across species:

Table 4: Comparative analysis of GMP synthase across different organisms

The conservation of the single polypeptide structure in Prochlorococcus marinus, as in other bacteria, suggests evolutionary pressure to maintain this organization for efficient nucleotide biosynthesis. The specific sequence adaptations in P. marinus likely reflect its evolution in nutrient-limited oceanic environments, where efficient nitrogen utilization is critical .

What insights can mutational studies provide about the catalytic mechanism of Prochlorococcus marinus GMP synthase?

Mutational studies of Prochlorococcus marinus GMP synthase can provide valuable insights into its catalytic mechanism and evolutionary adaptations. Based on sequence analysis, several approaches could be employed:

• Site-directed mutagenesis of predicted catalytic residues: Targeting conserved residues in both the glutamine amidotransferase domain and the synthetase domain to confirm their roles in catalysis.

• Domain interface mutations: Altering residues at the interface between domains to study interdomain communication and substrate channeling.

• Comparative mutational analysis: Creating mutations that convert P. marinus-specific residues to those found in homologs from other organisms to investigate adaptive evolution.

Table 5: Potential target residues for mutational studies based on sequence analysis

These mutational approaches, combined with enzyme kinetics and structural studies, can provide a comprehensive understanding of how Prochlorococcus marinus GMP synthase functions and how it has adapted to the organism's specific ecological niche .