Recombinant Staphylococcus aureus 3-hexulose-6-phosphate synthase (SAS0528)

What is 3-hexulose-6-phosphate synthase (HPS) and what reaction does it catalyze?

3-hexulose-6-phosphate synthase (EC 4.1.2.43, also known as D-arabino-3-hexulose 6-phosphate formaldehyde-lyase) is an enzyme with the systematic name D-arabino-hex-3-ulose-6-phosphate formaldehyde-lyase (D-ribulose-5-phosphate-forming). This enzyme catalyzes the following chemical reaction:

D-arabino-hex-3-ulose 6-phosphate → D-ribulose 5-phosphate + formaldehyde

The reaction is a key part of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation. Importantly, the enzyme requires Mg²⁺ or Mn²⁺ for maximal catalytic activity .

What are the optimal storage conditions for recombinant SAS0528?

The stability and shelf life of recombinant SAS0528 depend on several factors including storage state, buffer ingredients, and storage temperature. Based on experimental data:

• Liquid form has a shelf life of approximately 6 months when stored at -20°C or -80°C

• Lyophilized form maintains activity for up to 12 months at -20°C or -80°C

• Working aliquots can be stored at 4°C for up to one week

• Repeated freezing and thawing is not recommended as it may lead to protein denaturation and loss of activity

What is the recommended protocol for reconstitution of lyophilized SAS0528?

For optimal reconstitution of lyophilized SAS0528:

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

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation for long-term storage)

• Prepare small aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted aliquots at -20°C or -80°C for long-term storage

How can I verify the activity of recombinant SAS0528 in experimental settings?

The activity of recombinant SAS0528 can be assessed through multiple approaches:

• Spectrophotometric assay: Measure the formation of D-ribulose-5-phosphate following the reaction of SAS0528 with D-arabino-hex-3-ulose-6-phosphate

• Coupled enzyme assay: When studying the combined activity with PHI (6-phospho-3-hexuloisomerase), measure the formation of fructose-6-phosphate from formaldehyde and ribulose-5-phosphate. This assay has been used to determine activities of 172 U/mg for HPS-His₆ and 22 U/mg for the fused HPS-PHI protein

• Formaldehyde consumption: In engineered systems, measuring the rate of formaldehyde consumption can be used to verify SAS0528 activity. Escherichia coli strains expressing functional HPS show enhanced formaldehyde consumption compared to control strains

How does the structure of SAS0528 relate to its function?

The crystal structure of 3-hexulose-6-phosphate synthase from related organisms (e.g., Salmonella typhimurium at 1.65Å resolution) provides insights into the structure-function relationship of this enzyme family . Key structural features include:

• The protein adopts a homodimeric structure in its active form

• Each monomer contains a specific binding pocket for substrate recognition

• The active site includes metal-binding residues that coordinate Mg²⁺ or Mn²⁺, which are essential for catalytic activity

• The enzyme contains conserved residues that participate in formaldehyde addition to ribulose-5-phosphate

Understanding these structural elements is crucial for interpreting enzyme kinetics and designing experiments to modulate enzyme activity.

What is known about SAS0528's substrate specificity and kinetic parameters?

Research on 3-hexulose-6-phosphate synthase enzymes, including those from Staphylococcus aureus and other organisms, has revealed several important kinetic properties:

• Substrate specificity: The enzyme specifically catalyzes the formation of D-ribulose-5-phosphate and formaldehyde from D-arabino-hex-3-ulose-6-phosphate

• Catalytic efficiency: When comparing standalone HPS versus fused HPS-PHI proteins (from studies in other organisms like Methylomicrobium alcaliphilum), the k_cat/K_m ratio indicates that standalone HPS typically has higher catalytic efficiency but lower affinity for formaldehyde compared to HPS-PHI fusion proteins

• Inhibition: AMP and ADP have been identified as powerful inhibitors of both HPS and HPS-PHI activities, suggesting allosteric regulation mechanisms

How can SAS0528 be utilized in formaldehyde detoxification research?

SAS0528, as a 3-hexulose-6-phosphate synthase, plays a critical role in formaldehyde metabolism and can be applied in research focused on formaldehyde detoxification:

• Engineered detoxification systems: Expression of functional HPS (alone or as a fusion with PHI) in heterologous hosts has been shown to enhance formaldehyde consumption and improve growth in formaldehyde-containing media. For example, Escherichia coli strains harboring hps-phi fusion genes demonstrated increased resistance to formaldehyde toxicity

• Metabolic engineering: The enzyme can be incorporated into synthetic pathways to create organisms with enhanced ability to utilize or detoxify formaldehyde

• Environmental applications: Research into bioremediative processes for formaldehyde-contaminated environments can benefit from understanding and utilizing SAS0528's enzymatic properties

What experimental approaches can be used to study the interaction between SAS0528 and PHI?

Several methodologies have been employed to investigate the functional relationship between HPS and PHI:

• Recombinant expression: Generating constructs expressing HPS alone, PHI alone, or fused HPS-PHI to compare enzymatic activities

• Enzyme assays: Coupled enzyme assays measuring the sequential conversion of formaldehyde and ribulose-5-phosphate to fructose-6-phosphate

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation, yeast two-hybrid, or surface plasmon resonance to examine physical interactions

• Gene fusion experiments: Creating various fusion constructs (e.g., hps-phi and phi-hps) to analyze the effect of protein orientation on activity. Notably, studies with Mycobacterium gastri MB19 enzymes showed that hps-phi fusion exhibited both HPS and PHI activities, while phi-hps failed to display activity

How do fusion proteins of HPS and PHI compare to individual enzymes in terms of catalytic efficiency?

Research comparing individual HPS and PHI enzymes with their fusion proteins has revealed significant functional differences:

Studies with Methylomicrobium alcaliphilum 20Z enzymes showed that HPS-His₆ had activities of 172 U/mg, while the fused HPS-PHI protein exhibited 22 U/mg. Despite lower activity, the fusion protein often displayed higher substrate channeling efficiency between the two enzymatic domains .

What is the evolutionary significance of HPS and PHI gene organization across different species?

The genomic organization of HPS and PHI genes varies across bacterial species, providing insights into evolutionary adaptation:

• Separate genes: All RuMP pathway methylotrophs contain separately located hps and phi genes

• Fused genes: The hps-phi fused gene occurs only in several methanotrophs and is absent in methylotrophs not growing under methane

• Genomic context: Analysis of annotated genomes reveals that the hps-phi gene fusion may represent a specific adaptation in organisms that utilize methane as a carbon source

• Homologous recombination: In Staphylococcus aureus, there is evidence of homologous recombination between tandem gene paralogues, which drives the evolution of immunity gene clusters. While this specific mechanism hasn't been directly studied for hps genes, similar recombination processes might influence their evolution

What are the challenges in expressing and purifying functionally active SAS0528?

Researchers working with recombinant SAS0528 face several technical challenges:

• Expression system selection: Different expression systems (E. coli, mammalian, yeast, baculovirus) may yield proteins with varying activities and solubility. While E. coli is commonly used, mammalian or yeast expression systems might provide proteins with different post-translational modifications

• Protein solubility: Maintaining protein solubility during expression and purification can be challenging. The addition of solubility tags or optimization of buffer conditions may be necessary

• Maintaining enzymatic activity: The enzyme requires Mg²⁺ or Mn²⁺ for maximal activity, so appropriate metal ions must be included in buffers during purification and activity assays

• Protein stability: The protein has a limited shelf life, even under optimal storage conditions, necessitating careful planning of experiments and possibly fresh preparation of the enzyme for critical assays

How does SAS0528 compare structurally and functionally with HPS enzymes from other bacterial species?

Comparative analysis of HPS enzymes from different bacterial species reveals both conserved features and species-specific variations:

The crystal structure of HPS from Salmonella typhimurium (PDB: 3F4W) provides valuable structural information that can be used for homology modeling of SAS0528 .

What methods can be used to assess the impact of mutations on SAS0528 activity?

Several experimental approaches can be employed to evaluate how mutations affect SAS0528 activity:

• Site-directed mutagenesis: Targeted modification of specific amino acids to assess their role in catalysis or substrate binding

• Activity assays: Comparative kinetic analysis of wild-type and mutant enzymes using spectrophotometric or coupled enzyme assays

• Thermal stability analysis: Differential scanning fluorimetry or circular dichroism to assess changes in protein stability resulting from mutations

• Structural analysis: X-ray crystallography or molecular modeling to visualize structural perturbations caused by mutations

• In vivo complementation: Expressing mutant variants in formaldehyde-sensitive E. coli strains to assess functional restoration of formaldehyde resistance

How does SAS0528 function within the RuMP pathway for formaldehyde fixation?

SAS0528 plays a crucial role in the ribulose monophosphate (RuMP) pathway, which enables certain bacteria to fix formaldehyde into central metabolism:

Understanding this pathway integration is essential for metabolic engineering applications targeting formaldehyde utilization or detoxification.

What techniques can be used to study the metabolic flux through SAS0528 in vivo?

Several advanced methodologies can be employed to investigate the metabolic flux through SAS0528 in living cells:

• Isotope labeling: Using ¹³C-labeled formaldehyde to track carbon flux through the RuMP pathway

• Metabolomics analysis: Quantifying changes in metabolite levels (ribulose-5-phosphate, hexulose-6-phosphate, fructose-6-phosphate) in response to varying formaldehyde concentrations or enzyme expression levels

• In vivo enzyme assays: Permeabilized cell assays that maintain cellular context while allowing measurement of specific enzyme activities

• Reporter systems: Creating fusion proteins with fluorescent reporters to monitor protein expression and localization in real-time

• Genetic perturbations: Creating knockout or knockdown strains with altered HPS activity to assess pathway flux changes

These approaches collectively provide insights into how SAS0528 functions within its native metabolic context and how it might be harnessed for biotechnological applications.