Recombinant Photobacterium profundum Glucosamine--fructose-6-phosphate aminotransferase [isomerizing] (glmS), partial

What is Photobacterium profundum and why is it significant for studying glmS?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It is a Gram-negative rod-shaped bacterium with remarkable adaptability to various pressure and temperature conditions, capable of growth at temperatures ranging from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa depending on the strain . The organism was originally isolated from the Sulu Sea in 1986, with strain SS9 being particularly well-studied as a model piezophile . P. profundum's genome consists of two circular chromosomes and an 80 kb plasmid, and it shares close phylogenetic relationships with Vibrio species based on 16S rRNA sequence analysis .

The significance of P. profundum for studying glmS lies in its unique adaptations to high-pressure environments. The glmS gene encodes glucosamine-6-phosphate synthase, a critical enzyme that catalyzes the rate-limiting step in amino sugar biosynthesis, which is essential for bacterial cell wall formation . In P. profundum, this enzyme functions under extreme pressure conditions, making it an excellent model for understanding pressure adaptations of key metabolic enzymes. Furthermore, its ability to grow at both atmospheric and high pressures allows for comparative studies of enzyme function and regulation under different pressure regimes, providing insights into biochemical adaptations to deep-sea environments .

What are the optimal growth conditions for cultivating P. profundum strains for glmS research?

The cultivation of P. profundum strains requires specific conditions that replicate aspects of their natural deep-sea habitat while being practical for laboratory research. Different strains exhibit distinct optimal growth parameters that must be considered when designing experiments focused on glmS research. Strain SS9, the most extensively studied, grows optimally at 15°C and 28 MPa, exhibiting both psychrophilic and piezophilic characteristics . In contrast, strain 3TCK, isolated from San Diego Bay, thrives at 9°C and atmospheric pressure (0.1 MPa), while strain DSJ4, recovered from the Ryukyu Trench at a depth of 5110 m, performs best at 10°C and 10 MPa .

For laboratory cultivation, P. profundum requires salt, as it is a marine organism, and can metabolize various simple and complex carbohydrates . Growth can be monitored by measuring absorbance at 600 nm using microplate readers, which allows for high-throughput studies and statistical robustness through multiple replicates . When studying glmS expression specifically, researchers should consider that this organism's response to pressure involves changes in various metabolic pathways, with glycolysis/gluconeogenesis enzymes up-regulated at high pressure and oxidative phosphorylation proteins elevated at atmospheric pressure .

How does the glucosamine-6-phosphate synthase (glmS) function in bacterial metabolism?

Glucosamine-6-phosphate synthase, encoded by the glmS gene, performs a critical role in bacterial cell wall biosynthesis by catalyzing the formation of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-phosphate and glutamine. This reaction represents the first and rate-limiting step in the biosynthetic pathway leading to amino sugars that are essential components of bacterial cell walls . The enzyme exhibits remarkable catalytic properties, functioning both as an aminotransferase and isomerase to facilitate this complex conversion.

In bacterial metabolism, glmS serves as a crucial control point for cell wall synthesis, with its expression tightly regulated through feedback mechanisms. Specifically, the enzyme's product, GlcN-6-P, acts as a negative regulator of glmS expression at the post-transcriptional level . This elegant regulatory system ensures appropriate levels of amino sugar production based on cellular needs. When GlcN-6-P levels are low, glmS expression increases to restore adequate amino sugar synthesis; conversely, when GlcN-6-P concentrations are high, glmS expression is suppressed to prevent unnecessary resource expenditure .

The riboswitch functionality of glmS adds another layer of complexity to its regulation. The glmS mRNA contains a ribozyme domain that catalyzes self-cleavage when bound to GlcN-6-P, with the reaction occurring at the phosphodiester bond between residues A-1 and G1 through an acid-base mechanism involving the GlcN-6-P cofactor and G40 residue . This self-regulation mechanism represents one of nature's elegant solutions for metabolic control, directly linking enzyme production to substrate availability without requiring additional protein factors.

What specific adaptations does P. profundum glmS exhibit for functioning under high pressure?

At the molecular level, P. profundum responds to increased pressure by altering the composition of fatty acids in its cell membrane, suggesting that proteins like glmS may similarly feature structural modifications that maintain flexibility and function under compression . Pressure-adapted enzymes typically exhibit amino acid substitutions that favor smaller volume changes during catalysis, increased internal hydration, and modified electrostatic interactions that stabilize the active site under pressure.

The regulatory mechanisms controlling glmS expression also appear responsive to pressure conditions. The observation that glycolysis/gluconeogenesis pathway proteins are up-regulated at high pressure suggests that P. profundum prioritizes certain metabolic routes under pressure stress . Given glmS's critical role in cell wall biosynthesis, its expression and activity likely integrate into these pressure-responsive regulatory networks, ensuring that cell wall integrity and composition are maintained appropriately under varying pressure conditions.

How does the regulatory mechanism of glmS in P. profundum compare to that in other bacteria?

The regulation of glmS expression in bacteria involves sophisticated mechanisms that may vary across species. In Escherichia coli, glmS expression is controlled through a negative feedback loop wherein the enzyme's product, glucosamine-6-phosphate (GlcN-6-P), regulates gene expression at the post-transcriptional level . This regulatory system involves a small RNA called GlmZ (formerly RyiA or SraJ) that positively controls glmS mRNA levels in response to changes in intracellular GlcN-6-P concentration . When GlcN-6-P levels decrease, GlmZ accumulates in its unprocessed form, leading to increased glmS mRNA accumulation and, consequently, higher enzyme production .

In P. profundum, while the specific regulatory mechanism for glmS has not been explicitly detailed in the provided search results, we can infer potential adaptations based on the organism's pressure-responsive gene expression patterns. The bacterium modifies its protein expression profile significantly between atmospheric and high-pressure conditions, with different metabolic pathways being prioritized . Given that pressure affects metabolic pathway regulation, the glmS regulatory mechanism in P. profundum likely incorporates pressure-sensing components that may involve unique adaptations of the GlmZ-type system or entirely distinct mechanisms.

A critical factor in the regulation of glmS across bacterial species is the riboswitch functionality of its mRNA. The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1 through an acid-base mechanism involving the GlcN-6-P cofactor and G40 residue . This direct metabolite sensing represents an elegant solution for regulating enzyme production based on product availability. In P. profundum, this ribozyme mechanism may exhibit pressure-dependent kinetics or structural adaptations that optimize its function across the bacterium's wide pressure tolerance range.

What methodological approaches are most effective for studying P. profundum glmS under varying pressure conditions?

Investigating P. profundum glmS under different pressure conditions requires specialized methodologies that can maintain precise pressure control while allowing for analytical measurements. Several approaches have proven effective for such studies, with recent innovations enabling more accessible high-throughput analyses. A particularly valuable technique involves microplate-based growth monitoring under elevated hydrostatic pressure conditions, which allows for simultaneous tracking of multiple experimental conditions . This approach uses absorbance measurements at 600 nm to quantify bacterial growth, providing insights into how pressure affects cellular processes, including those dependent on glmS activity .

For direct protein analysis, shotgun proteomics with label-free quantitation and mass spectrometry has successfully identified differentially expressed proteins in P. profundum grown under atmospheric versus high-pressure conditions . This method can reveal changes in glmS abundance and modifications across pressure gradients. The technique involves:

• Culturing P. profundum at targeted pressure conditions (typically comparing 0.1 MPa and 28 MPa)

• Cell lysis and protein extraction under pressure-maintaining conditions

• Protein digestion and peptide separation

• Mass spectrometry analysis

• Bioinformatic processing to identify and quantify proteins

For investigating enzymatic activity under pressure, specialized high-pressure bioreactors equipped with spectrophotometric capabilities allow real-time monitoring of reaction kinetics. These systems can maintain pressures up to 100 MPa while permitting substrate addition and product sampling. When studying glmS specifically, researchers can track the conversion of fructose-6-phosphate to glucosamine-6-phosphate using coupled enzyme assays that generate spectrophotometrically detectable signals.

What are the catalytic mechanisms of P. profundum glmS ribozyme and how do they respond to pressure?

The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1 through a sophisticated acid-base mechanism that involves both the glucosamine-6-phosphate (GlcN-6-P) cofactor and the G40 residue . The current understanding of this catalytic process suggests that an external base first deprotonates either G40(N1) or possibly A-1(O2′), followed by proton transfer from G40(N1) to A-1(O2′) . After this initial deprotonation, A-1(O2′) attacks the phosphate as a hydroxyl group while hydrogen-bonded to the deprotonated G40, with G40(N1) directing the in-line attack . The proton transfer from A-1(O2′) to G40 occurs simultaneously with the attack on the scissile phosphate, completing the cleavage reaction .

Quantum mechanical/molecular mechanical free energy simulations and pKa calculations have indicated that catalysis is optimal when G40 has an elevated pKa rather than one shifted toward neutrality, although a balanced relationship among the pKa values of A-1, G40, and the nonbridging oxygen is essential for efficient catalysis . This delicate balance of acid-base properties within the active site is likely influenced by pressure conditions, as hydrostatic pressure can affect protonation states and hydrogen bonding networks.

While the specific effects of pressure on the P. profundum glmS ribozyme have not been directly addressed in the provided search results, we can infer potential impacts based on known pressure effects on biomolecular systems. High pressure typically compresses molecular structures, potentially altering the geometry of the ribozyme active site and affecting the positioning of catalytic residues. This compression could influence the pKa values of key groups, the strength of hydrogen bonds, and the rate of proton transfer steps, ultimately modifying the ribozyme's catalytic efficiency and regulatory function under deep-sea conditions.

How do the structural features of recombinant P. profundum glmS differ from those of mesophilic homologs?

Recombinant P. profundum glucosamine--fructose-6-phosphate aminotransferase [isomerizing] (glmS) likely exhibits distinct structural adaptations compared to mesophilic homologs, reflecting its evolution in a deep-sea, high-pressure environment. While the search results do not provide direct structural comparisons, we can infer potential differences based on P. profundum's piezophilic and psychrophilic characteristics. Proteins adapted to high-pressure environments often display several hallmark features: increased internal hydration, reduced cavity volumes, strengthened electrostatic interactions, and modifications that limit volume changes during conformational transitions .

The enzyme from P. profundum, particularly from strain SS9 which grows optimally at 28 MPa and 15°C, would be expected to maintain structural flexibility and catalytic efficiency under compression . This adaptation likely involves amino acid substitutions that favor smaller side chains at key positions, allowing the protein to undergo necessary conformational changes despite the volume-restricting effects of high pressure. Additionally, the enzyme would be expected to contain more charged residues on its surface to enhance solubility under pressure through electrostriction of water molecules.

Compared to mesophilic homologs such as the E. coli glmS enzyme, the P. profundum variant likely features a more flexible active site that remains accessible under pressure. This flexibility would be particularly important for accommodating the dual isomerase and aminotransferase functions required for converting fructose-6-phosphate to glucosamine-6-phosphate. The catalytic mechanism involving the important G40 residue, which appears essential based on mutation studies, may be preserved across homologs but with subtle adjustments in the P. profundum enzyme that optimize its function under deep-sea conditions .

What experimental approaches can assess the functional relationship between osmotic and hydrostatic pressure adaptations in P. profundum glmS?

Investigating the relationship between osmotic and hydrostatic pressure adaptations in P. profundum glmS requires integrated experimental approaches that can differentiate and correlate these two environmental stressors. This question is particularly relevant as physiological responses to increased hydrostatic pressure have been observed to parallel those triggered by osmotic pressure (increased salt concentration), with P. profundum SS9 producing similar intracellular osmolytes in response to both conditions .

A comprehensive experimental framework would include:

• Parallel pressure-osmolarity growth matrices: Cultivating P. profundum under combinations of hydrostatic pressure (0.1-90 MPa) and osmotic pressure (varying NaCl concentrations) while monitoring growth rates, cellular morphology, and glmS expression levels. This approach uses microplate readers with specialized pressure chambers to enable high-throughput analysis of multiple conditions simultaneously .

• Comparative proteomics and transcriptomics: Employing shotgun proteomics with label-free quantitation and mass spectrometry to identify differentially expressed proteins under various pressure and osmolarity conditions . Particular attention would be paid to glmS abundance and any post-translational modifications that might occur specifically under certain pressure-osmolarity combinations.

• Enzymatic activity assays under varying conditions: Measuring glmS catalytic efficiency and kinetic parameters across a matrix of hydrostatic and osmotic pressure values to determine how these stressors independently and synergistically affect enzyme function. This would involve spectrophotometric assays conducted in specialized high-pressure bioreactors.

• Metabolomics profiling: Quantifying intracellular osmolytes, amino sugars, and related metabolites under different pressure regimes to establish correlations between metabolic responses to hydrostatic versus osmotic pressure, with particular focus on glucosamine-6-phosphate levels and their relationship to glmS expression.

How can researchers optimize recombinant expression of P. profundum glmS in heterologous systems?

Achieving efficient heterologous expression of P. profundum glmS presents several challenges due to the enzyme's adaptation to high-pressure, low-temperature environments. Successful expression strategies must address codon usage differences, protein folding requirements, and potential toxicity issues. Based on general principles for expressing piezophilic proteins, the following methodological approach is recommended:

The initial step involves gene synthesis or amplification with codon optimization for the selected expression host, typically E. coli, though alternative hosts like Pseudomonas species may better accommodate certain features of marine bacterial proteins. Vector selection is critical, with pET-series vectors under the control of T7 promoters offering tight regulation and high expression potential . For challenging expressions, fusion tags such as SUMO, MBP, or GST can enhance solubility, with the tag positioned at the N-terminus to minimize interference with the enzyme's C-terminal catalytic residues.

Expression conditions significantly impact yield and activity of recombinant P. profundum glmS. Optimal results are typically achieved by cultivating host cells at reduced temperatures (15-20°C) following induction with lower IPTG concentrations (0.1-0.5 mM) than standard protocols. The growth medium should be supplemented with osmolytes such as glycine betaine (1-2 mM) to mimic aspects of the marine environment and stabilize protein folding. Cold-adapted chaperones may be co-expressed to facilitate proper folding of this psychrophilic enzyme.

Purification protocols must prioritize maintaining the native conformation of P. profundum glmS. All purification steps should be conducted at 4°C using buffers containing 10-15% glycerol and 300-400 mM NaCl to stabilize the enzyme. If high-pressure activity is a research focus, specialized equipment allowing protein purification under elevated pressure conditions should be employed. Activity verification using spectrophotometric assays that monitor the conversion of fructose-6-phosphate to glucosamine-6-phosphate is essential to confirm that the recombinant enzyme retains its catalytic properties.

What analytical techniques can determine pressure-induced conformational changes in the glmS enzyme?

Investigating pressure-induced conformational changes in the glmS enzyme requires specialized analytical techniques that can operate under high-pressure conditions or capture structural states that persist after pressure treatment. Several complementary approaches provide insights into different aspects of pressure-induced protein alterations:

High-pressure nuclear magnetic resonance (HP-NMR) spectroscopy represents one of the most powerful techniques for directly monitoring protein conformational changes under pressure. This approach can detect alterations in chemical shifts that reflect changes in the chemical environment of specific amino acid residues as the protein structure responds to compression. For glmS enzyme studies, HP-NMR can identify which regions of the protein exhibit the greatest structural flexibility under pressure and potentially correlate these changes with catalytic activity . The technique requires isotopically labeled protein (typically with 15N, 13C, or 2H) and specialized high-pressure NMR cells capable of withstanding pressures up to 200 MPa.

Fluorescence spectroscopy under pressure offers another direct measurement approach, particularly when combined with site-specific fluorescent labeling of the glmS enzyme. Intrinsic tryptophan fluorescence can monitor global conformational changes, while strategically placed extrinsic fluorophores can report on local structural alterations in specific domains. Changes in fluorescence intensity, emission maximum wavelength, and anisotropy provide information about solvent exposure, polarity changes, and rotational mobility of different protein regions under pressure.

X-ray crystallography combined with high-pressure cryo-cooling techniques can capture pressure-induced structural states for detailed atomic-level analysis. This approach involves subjecting protein crystals to high pressure during flash-cooling, trapping conformational states that can then be analyzed by standard crystallographic methods. For glmS, this could reveal pressure-induced alterations in the active site geometry that might explain any observed changes in catalytic properties.

How can researchers investigate the regulatory network controlling glmS expression in P. profundum?

Investigating the regulatory network controlling glmS expression in P. profundum requires a multi-faceted approach that integrates genomic, transcriptomic, and biochemical methodologies. The complexity of this network, which likely incorporates both pressure-responsive elements and metabolite-sensing mechanisms, necessitates systematic experimental strategies to fully elucidate its components and interactions.

Transcriptome analysis using RNA sequencing (RNA-seq) under varying pressure conditions provides a comprehensive view of how glmS expression changes in response to environmental pressure. This approach should be conducted by comparing P. profundum cultures grown at atmospheric pressure (0.1 MPa) versus optimal pressure (28 MPa) and stress-inducing high pressure (70-90 MPa) . Time-course analyses during pressure transitions are particularly valuable for identifying early regulatory responses. The detection of small regulatory RNAs analogous to E. coli's GlmZ should be prioritized, as these may play crucial roles in post-transcriptional regulation of glmS .

Genomic and promoter analysis techniques provide insights into the cis-regulatory elements controlling glmS expression. Researchers should construct reporter gene fusions using the glmS promoter and upstream regions linked to fluorescent proteins or luciferase to monitor expression in vivo under different pressure conditions. Site-directed mutagenesis of potential regulatory sites in the promoter region can identify specific pressure-responsive elements. Additionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) can identify transcription factors that bind to the glmS promoter region under various pressure conditions.

Biochemical characterization of the glmS ribozyme's pressure response provides direct insights into post-transcriptional regulation. In vitro transcription of the glmS mRNA followed by self-cleavage assays conducted under varying pressure conditions can reveal how pressure affects the ribozyme's catalytic activity. The binding affinity of glucosamine-6-phosphate to the ribozyme can be measured using isothermal titration calorimetry at different pressures to determine if pressure alters the metabolite-sensing capability of the ribozyme.

How can the study of P. profundum glmS contribute to our understanding of deep-sea microbial adaptations?

The study of P. profundum glmS offers a unique window into how essential metabolic enzymes adapt to function in extreme deep-sea environments. As a key enzyme in cell wall biosynthesis, glmS represents a critical link between central carbon metabolism and cell structure, making its adaptation to high-pressure environments particularly significant for understanding how deep-sea microbes maintain cellular integrity and growth under extreme conditions. The pressure adaptations observed in P. profundum can serve as a model for understanding broader principles of deep-sea microbial evolution and biochemical adaptation.

One particularly valuable aspect of studying P. profundum glmS is the opportunity to investigate the molecular basis of pressure adaptation in an enzyme with well-characterized homologs from mesophilic organisms. By comparing the structure, function, and regulation of glmS across P. profundum strains adapted to different pressure regimes (from atmospheric pressure to 70 MPa), researchers can identify specific amino acid substitutions and structural modifications that confer pressure tolerance . These comparative studies can reveal whether pressure adaptation occurs through common mechanisms across diverse proteins or involves enzyme-specific solutions.

The dual stress response of P. profundum to both hydrostatic pressure and osmotic pressure provides insights into potential evolutionary relationships between these environmental challenges . The observation that P. profundum produces similar intracellular osmolytes in response to both salt and hydrostatic pressure suggests potential mechanistic overlaps in cellular responses to these stressors . Understanding how glmS regulation integrates into these broader stress response networks offers a systems-level perspective on deep-sea adaptation, potentially revealing how ancient marine organisms evolved to colonize the extreme environments of the deep ocean.

What are the implications of pressure-adaptive features of glmS for biocatalysis and biotechnology?

The pressure-adaptive features of P. profundum glmS have significant implications for biocatalysis and biotechnology, particularly in the development of enzymes capable of functioning under non-standard conditions. By understanding how this enzyme maintains catalytic efficiency under high pressure, researchers can potentially engineer novel biocatalysts with enhanced stability and activity for industrial processes that operate under extreme conditions. These applications span multiple biotechnological sectors, from pharmaceutical manufacturing to food processing.

High-pressure biocatalysis offers several advantages for industrial applications, including increased reaction rates, altered substrate specificity, and reduced risk of microbial contamination. Enzymes like P. profundum glmS that naturally function under pressure can serve as templates for designing biocatalysts specifically tailored for high-pressure bioprocessing. The amino acid substitutions and structural features that confer pressure tolerance to glmS could be transferred to other industrially relevant enzymes, creating chimeric proteins with enhanced pressure stability while maintaining desired catalytic properties.

The glmS ribozyme component presents particularly intriguing biotechnological applications as a pressure-responsive genetic regulatory element. By incorporating the pressure-adapted glmS ribozyme into synthetic genetic circuits, researchers could develop biosensors that respond specifically to pressure changes or create expression systems that are activated only under specific pressure conditions . Such pressure-regulated gene expression systems could have applications in contained bioremediation, where engineered organisms would become metabolically active only under defined pressure conditions, providing an additional layer of biocontainment.

How might climate change and ocean acidification affect P. profundum glmS function in deep-sea environments?

Climate change and ocean acidification present significant challenges to deep-sea ecosystems, with potential implications for the function of P. profundum glmS and similar enzymes in piezophilic microorganisms. The deep ocean is experiencing gradual warming, deoxygenation, and acidification due to increased atmospheric CO2 levels, creating multiple stressors that may impact enzyme function and microbial metabolism in ways that remain poorly understood. Understanding these effects requires integrating knowledge of enzyme biochemistry with environmental change projections.

Ocean acidification, resulting from increased dissolution of atmospheric CO2, leads to decreased pH in marine environments. This pH change may directly affect glmS enzymatic activity by altering the protonation states of catalytic residues, particularly affecting the acid-base chemistry that is critical for the ribozyme function . The complex catalytic mechanism involving proton transfers and specific pKa values for key residues in the active site may be particularly sensitive to environmental pH changes. Additionally, ocean acidification could impact the availability of divalent metal ions essential for ribozyme function, potentially affecting glmS regulatory capabilities.

Temperature increases in deep-sea environments, while subtle compared to surface warming, may significantly impact psychrophilic enzymes like those from P. profundum. The thermal stability of these cold-adapted proteins is typically lower than that of mesophilic homologs, making them potentially vulnerable to even small increases in environmental temperature. For P. profundum, which exhibits optimal growth at 15°C (strain SS9), warming could push enzymatic systems beyond their evolutionary adaptations . The combined effects of temperature and pressure on protein stability and function are complex and non-additive, suggesting that climate change could disrupt the delicate balance of adaptations that allow enzymes like glmS to function efficiently in the deep sea.