Recombinant Photobacterium profundum GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is Photobacterium profundum GMP synthase and what is its function?

GMP synthase [glutamine-hydrolyzing] (guaA, EC 6.3.5.2) from Photobacterium profundum SS9 is a bifunctional enzyme that catalyzes the final step in de novo GMP biosynthesis. This enzyme converts XMP (xanthosine monophosphate) to GMP using glutamine as an amide donor. In P. profundum, this protein is encoded by the guaA gene (PBPRA0781) and consists of 527 amino acids . The protein plays a critical role in purine nucleotide biosynthesis, which is essential for DNA and RNA synthesis, as well as for various signaling pathways.

The enzyme is bifunctional, containing both GMP synthase and glutamine amidotransferase activities, allowing it to catalyze the ATP-dependent amidation of XMP to GMP. This bifunctionality is reflected in its structural organization, with distinct domains responsible for each activity. The protein's structure has been computationally modeled with high confidence (pLDDT global score of 90.9), suggesting a well-ordered tertiary structure .

What are the optimal growth conditions for Photobacterium profundum SS9 prior to recombinant protein expression?

P. profundum SS9 is a piezophilic (pressure-loving) bacterium that grows optimally at 28 MPa (approximately 280 atmospheres) and 15°C . Laboratory culture of this organism typically employs marine broth (28 g/liter 2216 medium) supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5) . For anaerobic growth conditions, cultures are often grown in sealed containers with minimal headspace to maintain pressure uniformity.

Standard laboratory protocols include:

• Inoculation from -80°C freezer stocks into marine broth

• Growth at 15-17°C until reaching appropriate optical density

• Transfer to pressure vessels for high-pressure growth when studying pressure-responsive phenotypes

Importantly, while P. profundum grows optimally at high pressure, it can also grow at atmospheric pressure, facilitating genetic manipulation and routine laboratory culture. This adaptability makes it an excellent model organism for studying piezophily .

How is the guaA gene organized in the P. profundum SS9 genome?

The guaA gene in P. profundum SS9 is located on chromosome 1 at position 864532..866115 in the genome, encoding a protein of 527 amino acids . The gene is flanked by:

• Upstream: An inositol-5-monophosphate dehydrogenase gene (position 862936..864399)

• Downstream: A putative p-aminobenzoyl-glutamate transporter gene (position 866566..868125)

This genomic organization suggests potential co-regulation or functional relationships between guaA and these neighboring genes. The guaA gene is specifically identified as PBPRA0781 in the genome annotation, with a GC content of approximately 44.51%, which is within the typical range for P. profundum genes .

What expression systems are suitable for recombinant production of P. profundum guaA?

While the search results don't specifically address expression systems for P. profundum guaA, standard recombinant protein expression approaches can be adapted. Based on methodologies used with other P. profundum proteins, the following systems would likely be suitable:

• E. coli-based expression systems:

  • BL21(DE3) strains for T7-based expression

  • Arctic Express strains for cold-adapted expression (relevant given P. profundum's psychrophilic nature)

  • Rosetta strains to address codon usage differences between E. coli and P. profundum

• Cold-adapted expression protocols:

  • Induction at lower temperatures (15-20°C)

  • Extended expression periods (24-48 hours)

  • Use of mild inducers (0.1-0.5 mM IPTG)

• Specialized conditions:

  • Inclusion of osmolytes or pressure-mimicking conditions

  • Marine-based media supplements

These approaches take into account P. profundum's adaptation to cold, high-pressure environments while utilizing standard laboratory expression hosts.

How does pressure affect the expression and activity of P. profundum GMP synthase?

Proteomic studies have shown that P. profundum differentially expresses numerous proteins in response to varying hydrostatic pressure conditions. While the search results don't specifically mention pressure effects on GMP synthase, related metabolic pathways show clear pressure-dependent regulation patterns. Glycolysis/gluconeogenesis pathway proteins are typically up-regulated at high pressure (28 MPa), while oxidative phosphorylation pathway components tend to be up-regulated at atmospheric pressure .

Given that GMP synthase participates in nucleotide metabolism, which interfaces with central carbon metabolism, its expression and activity may similarly be pressure-responsive. Researchers investigating pressure effects specifically on GMP synthase should consider:

• Comparative expression analysis at different pressures (0.1 MPa vs. 28 MPa)

• Enzyme kinetics studies under varying pressure conditions

• Structural stability assessments at different pressures

• Potential adaptation of the active site for function under high pressure

The protein's structure, modeled with high confidence (pLDDT global score: 90.9) , might contain features that facilitate function under pressure, though specific pressure-adapted structural elements would require experimental verification.

What purification strategies are most effective for recombinant P. profundum GMP synthase?

Based on general principles for purifying GMP synthase and the characteristics of proteins from extremophiles like P. profundum, the following purification strategy would be recommended:

Table 1: Recommended Purification Protocol for Recombinant P. profundum GMP synthase

This purification scheme incorporates:

• Cold temperature maintenance throughout (4-8°C)

• Increased salt concentration to maintain stability of halophilic proteins

• Use of glycerol as a stabilizing agent

• DTT to maintain reduced state of cysteine residues

Researchers should verify final purity by SDS-PAGE and confirm activity using appropriate enzyme assays.

How can the enzymatic activity of P. profundum GMP synthase be reliably measured?

The enzymatic activity of GMP synthase can be measured through several complementary approaches:

• Spectrophotometric assay: Monitoring the decrease in absorbance at 290 nm, corresponding to the conversion of XMP to GMP. This can be performed in a reaction buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 100 mM KCl

  • 5 mM MgCl₂

  • 1 mM ATP

  • 1 mM glutamine

  • 0.1 mM XMP

  • 1 mM DTT

• HPLC-based assay: Quantifying the production of GMP directly by separating reaction products on a C18 column using appropriate mobile phases and UV detection at 252 nm.

• Coupled enzyme assay: Using auxiliary enzymes to couple GMP production to NADH oxidation, which can be monitored spectrophotometrically at 340 nm.

Important considerations specific to P. profundum GMP synthase include:

• Temperature effects (assays at 15°C vs. standard temperatures)

• Pressure effects (specialized high-pressure enzyme assay chambers may be required)

• Salt concentration optimization (given the marine origin of the organism)

How do the structural and functional properties of P. profundum GMP synthase compare to homologs from non-piezophilic bacteria?

P. profundum GMP synthase shares structural similarities with homologs from other bacteria, but may possess unique adaptations related to high-pressure environments. The AlphaFold computational model (AF-Q6LU31-F1) released in 2021 and modified in 2022 shows a high-confidence prediction of the protein's structure (global pLDDT score of 90.9) .

Comparative analyses should focus on:

Table 2: Key Structural Comparisons Between Piezophilic and Non-Piezophilic GMP Synthases

While specific experimental data comparing these features is not provided in the search results, researchers typically observe these patterns when comparing enzymes from piezophilic organisms to mesophilic counterparts. Advanced structural analyses using X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, or molecular dynamics simulations would be necessary to confirm these hypothesized differences.

What genetic manipulation techniques are most effective for studying P. profundum guaA function in vivo?

Based on established methods for P. profundum genetic manipulation described in the search results, the following techniques would be effective for studying guaA function:

• Conjugal delivery of plasmids: Bi-parental mating between E. coli (containing appropriate vectors) and P. profundum strains has been successfully used for genetic transfer . This approach allows for introduction of plasmids carrying wild-type or modified guaA genes.

• Transposon mutagenesis: Large-scale transposon mutagenesis has been employed for P. profundum, including with Tn5-B30 . This approach could generate guaA mutants or mutants in related pathways.

• Gene disruption methods: Targeted gene disruption can be accomplished using internal gene fragments cloned into suicide vectors (like pMUT100) that integrate into the chromosome via single-crossover events . For guaA, primers could be designed to amplify internal fragments for such disruption.

• Plasmid rescue techniques: For identifying and characterizing genetic regions, plasmid rescue approaches have been successfully applied, where genomic DNA is digested, circularized, and transformed into E. coli .

The methodology typically involves:

• Culture incubation at 15-17°C

• Use of marine broth supplemented with appropriate antibiotics

• Mating procedures on membrane filters

• Selection on appropriate media

• Verification of genetic modifications by PCR and sequencing

These approaches could be applied to study guaA function through complementation of mutants, overexpression studies, or expression of modified versions of the gene.

How might the pressure-adapted properties of P. profundum GMP synthase be leveraged for biotechnological applications?

While the search results don't directly address biotechnological applications of P. profundum GMP synthase, several potential applications can be inferred based on its pressure-adapted properties:

• Biocatalysis under non-conventional conditions:

  • High-pressure enzymatic processes (100-300 MPa)

  • Low-temperature bioprocessing (5-15°C)

  • Combined pressure-temperature bioprocessing

• Structural biology insights:

  • Template for engineering pressure-stable enzymes

  • Model for understanding protein adaptation to extreme conditions

  • Development of pressure-resistant protein scaffolds

• Nucleotide synthesis applications:

  • Production of modified nucleotides under pressure conditions

  • Pressure-enhanced selectivity for certain substrates

  • Cold-adapted nucleotide synthesis processes

• Pharmaceutical and research reagent applications:

  • Development of pressure-stable enzyme formulations

  • Cold-active enzyme reagents for molecular biology

  • Novel substrate specificities for nucleotide analog synthesis

These applications would require thorough characterization of the enzyme's kinetic parameters under various pressure conditions, as well as engineering efforts to optimize expression, stability, and catalytic properties for specific biotechnological purposes.

What methods can be used to investigate the regulatory mechanisms controlling P. profundum guaA expression under different pressure conditions?

To investigate pressure-responsive regulation of guaA expression in P. profundum, researchers could employ several complementary approaches:

• Transcriptomic analysis:

  • RNA-Seq comparing expression at different pressures (0.1 MPa vs. 28 MPa)

  • qRT-PCR validation of guaA expression changes

  • Transcription start site mapping using 5' RACE

• Promoter analysis:

  • Identification of regulatory elements in the guaA promoter region

  • Reporter gene assays using promoter-reporter fusions

  • DNA-protein interaction studies (EMSA, ChIP-seq) to identify trans-acting factors

• Proteomic approaches:

  • Label-free quantitation mass spectrometry (as described in search result 3)

  • Targeted proteomics focusing on GMP synthase and related proteins

  • Protein stability and turnover studies under different pressure conditions

• Genetic approaches:

  • Construction of promoter deletion/mutation series

  • Identification of regulatory mutants with altered pressure response

  • Transposon mutagenesis screening for regulators of guaA expression

Table 3: Experimental Design for Investigating Pressure-Responsive guaA Regulation

The culture methodology would follow established protocols for P. profundum growth at different pressures, as described in search result 3, using pressure vessels capable of maintaining controlled hydrostatic pressure conditions .

What are common challenges in recombinant expression of P. profundum guaA and how can they be addressed?

Recombinant expression of proteins from extremophiles like P. profundum often presents unique challenges. For guaA specifically, researchers might encounter:

• Protein solubility issues:

  • Problem: Formation of inclusion bodies due to improper folding in mesophilic expression hosts

  • Solution: Lower induction temperature (15-18°C), reduce inducer concentration, use solubility-enhancing fusion tags (SUMO, MBP), or add osmolytes to the growth medium

• Low expression levels:

  • Problem: Codon usage differences between P. profundum and expression host

  • Solution: Use codon-optimized synthetic gene, employ Rosetta strains containing rare tRNAs, or optimize ribosome binding sites

• Loss of activity during purification:

  • Problem: Protein destabilization during pressure transition

  • Solution: Include stabilizing agents (glycerol, specific ions), maintain cold temperatures throughout purification, consider rapid purification protocols

• Difficulty in reproducing native conditions:

  • Problem: Standard lab conditions don't reflect the high-pressure, low-temperature native environment

  • Solution: Specialized high-pressure expression systems or post-expression pressure treatment of purified protein

How can researchers distinguish between pressure effects on GMP synthase structure versus effects on its catalytic mechanism?

Differentiating between pressure effects on protein structure versus catalytic mechanism requires a multi-faceted experimental approach:

• Structural analysis under pressure:

  • High-pressure X-ray crystallography

  • High-pressure NMR spectroscopy

  • High-pressure circular dichroism

  • Molecular dynamics simulations under varying pressure conditions

• Catalytic mechanism investigation:

  • Pre-steady-state kinetics under varying pressures

  • Isotope effect studies at different pressures

  • Pressure dependence of individual catalytic steps

  • Site-directed mutagenesis of catalytic residues followed by pressure-dependent activity assays

• Hybrid approaches:

  • Hydrogen-deuterium exchange mass spectrometry at varying pressures

  • Trapping catalytic intermediates under pressure

  • Temperature-pressure phase diagrams of activity versus structural stability

By comparing the pressure dependence of structural parameters (measured by spectroscopic methods) with the pressure dependence of catalytic parameters (measured by kinetic methods), researchers can distinguish between effects on structure versus catalysis. Volume changes associated with specific catalytic steps can be particularly informative in understanding pressure effects on enzyme mechanisms.

What strategies can address reproducibility challenges when working with pressure-adapted enzymes like P. profundum GMP synthase?

Ensuring reproducible results when working with pressure-adapted enzymes presents several unique challenges. Recommended strategies include:

• Standardized pressure treatment protocols:

  • Use calibrated pressure equipment with precise control

  • Document pressure ramping rates and holding times

  • Maintain consistent temperature during pressure treatment

  • Include pressure-stable control samples in experiments

• Protein preparation consistency:

  • Standardize expression conditions (particularly temperature and induction parameters)

  • Establish rigorous purification protocols with quality control checkpoints

  • Characterize protein batches (circular dichroism, thermal stability, activity assays)

  • Store samples with pressure-protective additives (glycerol, specific salts)

• Experimental design considerations:

  • Include technical and biological replicates

  • Perform pressure cycling experiments to assess reversibility

  • Use internal standards appropriate for high-pressure experiments

  • Document pre-experimental history of protein samples

• Data analysis and reporting:

  • Apply appropriate statistical tests for pressure-dependent data

  • Report detailed methodological parameters in publications

  • Share raw data to enable meta-analysis

  • Use consistent data normalization approaches

By implementing these strategies, researchers can improve reproducibility and enable meaningful comparison of results across different laboratories studying pressure-adapted enzymes.

What are the most promising future research directions for P. profundum GMP synthase?

Based on the current state of knowledge reflected in the search results, several promising research directions emerge:

• Structural biology under pressure:

  • Determination of high-resolution crystal structure under various pressure conditions

  • Comparison with mesophilic homologs to identify pressure-adaptation features

  • Investigation of pressure-dependent conformational changes using spectroscopic methods

• Metabolic integration:

  • Systems biology approach to understand nucleotide metabolism under pressure

  • Metabolomics studies of guanine nucleotide pools at different pressures

  • Investigation of potential moonlighting functions under pressure stress

• Evolutionary aspects:

  • Comparative genomics across piezophilic bacteria

  • Ancestral sequence reconstruction and functional characterization

  • Identification of convergent adaptations to high pressure

• Biotechnological applications:

  • Engineering pressure-stable variants with enhanced catalytic properties

  • Development of high-pressure biocatalytic processes

  • Exploration of substrate promiscuity for synthesis of modified nucleotides

These research directions build upon the foundation of knowledge about P. profundum biology, particularly its adaptation to high-pressure environments, while expanding into new areas with both fundamental and applied significance.