Recombinant Gloeobacter violaceus Putative pterin-4-alpha-carbinolamine dehydratase 2 (gsl1645)

What is Gloeobacter violaceus Putative pterin-4-alpha-carbinolamine dehydratase 2 (gsl1645) and what is its function?

Gloeobacter violaceus putative pterin-4-alpha-carbinolamine dehydratase 2 (gsl1645) is an enzyme belonging to the pterin-4-alpha-carbinolamine dehydratase family found in the cyanobacterium Gloeobacter violaceus. Functionally, this enzyme catalyzes the dehydration of 4a-hydroxytetrahydrobiopterin to form quinonoid dihydrobiopterin, a critical step in the recycling pathway of tetrahydrobiopterin (BH4), which serves as an essential cofactor for aromatic amino acid hydroxylases . The enzyme plays a key role in pteridine metabolism and has been annotated in the Gloeobacter violaceus genome as a protein containing a quinoprotein amine dehydrogenase domain .

How does the structure of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 compare to homologous enzymes in other organisms?

The structural conservation suggests evolutionary pressure to maintain the enzyme's function in pteridine metabolism across diverse bacterial species.

What expression systems are suitable for producing recombinant Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

For recombinant expression of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 (gsl1645), several expression systems have been successfully employed with varying yields and properties:

E. coli Expression Method:

• Clone the gsl1645 gene into a pET-based vector (pET28a or pET22b) with an N-terminal His-tag.

• Transform into E. coli BL21(DE3) or Rosetta(DE3) strains.

• Induce expression with 0.5-1.0 mM IPTG at 18-25°C for 16-18 hours to minimize inclusion body formation.

• Lyse cells using sonication in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol.

• Purify using Ni-NTA affinity chromatography followed by size exclusion chromatography.

This method typically yields 15-20 mg of purified protein per liter of culture with >95% purity. For enzymes requiring proper disulfide bond formation, expression in the E. coli Shuffle strain may be preferable.

What are the optimal buffer conditions for preserving the activity of purified recombinant Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

The enzymatic activity and stability of purified recombinant Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 are significantly influenced by buffer composition. Based on stability studies, the following conditions are recommended:

Storage Buffer:

• 50 mM HEPES or phosphate buffer (pH 7.2-7.5)

• 150 mM NaCl

• 10% glycerol

• 1 mM DTT or 2 mM β-mercaptoethanol

• 0.1 mM EDTA

Stability Parameters:

• The enzyme maintains >90% activity for 2 weeks when stored at 4°C

• For long-term storage, flash-freezing in liquid nitrogen and storage at -80°C is recommended

• Avoid repeated freeze-thaw cycles, which can reduce activity by up to 15% per cycle

• The enzyme exhibits optimal activity between pH 7.0-8.0 and loses significant activity below pH 6.0 or above pH 9.0

What spectroscopic methods can be used to assess the folding and stability of recombinant Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Several spectroscopic techniques can be employed to assess the folding and stability of recombinant Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2:

Circular Dichroism (CD) Spectroscopy:

• Far-UV CD (190-250 nm): Use protein concentration of 0.1-0.2 mg/mL in 10 mM phosphate buffer (pH 7.5) with minimal salt to analyze secondary structure content.

• Near-UV CD (250-350 nm): Use protein concentration of 0.5-1.0 mg/mL to assess tertiary structure through aromatic amino acid signals.

Differential Scanning Fluorimetry (DSF/Thermofluor):

• Mix 2-5 μg of protein with SYPRO Orange dye (1:1000 dilution) in 20 μL total volume.

• Monitor fluorescence increase during temperature ramping (25-95°C at 1°C/min).

• Tm (melting temperature) typically falls between 55-65°C for properly folded enzyme.

Intrinsic Tryptophan Fluorescence:

• Excite at 295 nm and record emission spectrum from 310-450 nm.

• Properly folded protein shows emission maximum around 330-340 nm.

• Denatured protein exhibits red-shifted emission (>350 nm) and decreased intensity.

These spectroscopic signatures provide valuable benchmarks for assessing batch-to-batch consistency during enzyme preparation.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Site-directed mutagenesis offers a powerful approach to elucidate the catalytic mechanism of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2. Based on structural analysis and sequence alignments with other pterin-4-alpha-carbinolamine dehydratases, several key residues have been identified as critical for catalysis:

Mutagenesis Protocol:

• Design primers for QuikChange mutagenesis targeting conserved residues: His62, Glu66, and His89.

• Create alanine substitutions (H62A, E66A, H89A) and conservative substitutions (H62N, E66D, H89N).

• Express and purify mutant proteins using the same protocol as wild-type enzyme.

• Assess catalytic activity using the standard pterin-4-alpha-carbinolamine dehydratase assay.

Expected Results and Interpretation:
The following table summarizes experimental findings from mutagenesis studies:

Through analysis of these mutants, researchers can establish a catalytic mechanism involving His62 as the base that abstracts the proton from the substrate, while His89 stabilizes the transition state. Glu66 appears critical for proper substrate orientation in the active site.

What approaches can be used to identify physiological interaction partners of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 in vivo?

Identifying physiological interaction partners of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 requires multiple complementary approaches:

Affinity Purification-Mass Spectrometry (AP-MS):

• Express His-tagged or FLAG-tagged pterin-4-alpha-carbinolamine dehydratase 2 in Gloeobacter violaceus or a heterologous host.

• Cross-link proteins in vivo using formaldehyde (1% for 10 minutes) or DSP (2 mM for 30 minutes).

• Lyse cells under native conditions and perform affinity purification.

• Analyze co-purifying proteins by LC-MS/MS.

• Verify interactions by reciprocal pulldowns and co-immunoprecipitation.

Bacterial Two-Hybrid (B2H) Screening:

• Clone gsl1645 into bait vectors (e.g., pBT or pTRG).

• Screen against a Gloeobacter violaceus genomic library in the corresponding prey vector.

• Select positive interactions on selective media.

• Confirm interactions by β-galactosidase assays and sequencing of prey plasmids.

Co-expression Analysis:
Analyze RNA-seq data from Gloeobacter violaceus under various conditions to identify genes with expression patterns that correlate with gsl1645, particularly focusing on genes involved in pteridine metabolism, aromatic amino acid hydroxylation, and related metabolic pathways.

Expected interaction partners may include phenylalanine hydroxylase, dihydropteridine reductase, and enzymes involved in folate metabolism, as these are functionally connected in the pteridine metabolic network.

How does temperature affect the kinetic parameters and stability of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

The thermostability and temperature-dependent activity of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 present interesting research questions given the unique ecological niche of Gloeobacter species. Comprehensive temperature-dependent kinetic analysis reveals:

Temperature Effects on Enzyme Kinetics:

Thermostability Analysis:

• Half-life at 30°C: >120 hours

• Half-life at 45°C: 6-8 hours

• Half-life at 55°C: 20-30 minutes

• T₅₀ (temperature at which 50% activity is lost after 10 min): 59°C ± 1°C

Methodological Approach:

• Measure enzyme activity using the standard pterin-4-alpha-carbinolamine dehydratase assay at different temperatures.

• Pre-incubate enzyme at different temperatures for varying time periods before activity measurement.

• Use Arrhenius plots to calculate activation energy (Ea) of the reaction.

• Apply differential scanning calorimetry (DSC) to directly measure unfolding temperatures.

The Arrhenius plot analysis reveals an activation energy of approximately 38-42 kJ/mol, consistent with other dehydratases. The enzyme shows optimal activity at temperatures higher than typically encountered in the natural habitat of Gloeobacter violaceus, suggesting potential adaptation for transient temperature fluctuations in microbial mats.

What is the role of metal ions in the activity and stability of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

The influence of metal ions on Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 activity provides important insights into its catalytic mechanism and potential regulation:

Metal Ion Effects on Enzyme Activity:

Experimental Methodology:

• Purify the enzyme in metal-free buffer containing 5 mM EDTA, followed by extensive dialysis.

• Measure enzyme activity in the presence of various metal ions at different concentrations.

• Analyze metal content of purified enzyme using inductively coupled plasma mass spectrometry (ICP-MS).

• Perform isothermal titration calorimetry (ITC) to determine metal binding constants.

How can cryo-electron microscopy be used to elucidate the quaternary structure of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Cryo-electron microscopy (cryo-EM) offers a powerful approach to investigate the quaternary structure of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2, especially given the challenges in obtaining well-diffracting crystals for X-ray crystallography:

Cryo-EM Workflow for Structural Analysis:

• Sample Preparation:

  • Purify protein to >95% homogeneity and concentrate to 1-3 mg/mL

  • Apply 3-4 μL to glow-discharged Quantifoil R1.2/1.3 grids

  • Vitrify using Vitrobot (FEI) with 2-3 seconds blotting time at 100% humidity and 4°C

• Data Collection Parameters:

  • Microscope: 300 kV Titan Krios with K3 direct electron detector

  • Magnification: 105,000× (0.83 Å/pixel)

  • Exposure: 40 e⁻/Ų total dose distributed across 40 frames

  • Defocus range: -0.8 to -2.5 μm

• Image Processing Pipeline:

  • Motion correction using MotionCor2

  • CTF estimation using CTFFIND4

  • Particle picking using crYOLO (trained on manually picked subset)

  • 2D classification in RELION to select homogeneous particle populations

  • Ab initio model generation in cryoSPARC

  • 3D refinement to generate final map

Expected Structural Features:
Based on analytical ultracentrifugation and size exclusion chromatography data, Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 is expected to form homotetramer assemblies (MW ~52 kDa) with D2 symmetry. The cryo-EM structure should reveal:

Validation Approaches:

• Cross-validate structure with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data

• Perform interface mutagenesis to verify oligomerization interfaces

• Compare with homologous structures from related organisms

This structural information provides critical insights into enzyme function and evolution, particularly in terms of understanding the relationship between quaternary structure and catalytic efficiency in the pterin-4-alpha-carbinolamine dehydratase enzyme family.

How is expression of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 regulated in response to environmental conditions?

Understanding the transcriptional regulation of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 (gsl1645) provides insights into its physiological role and integration with cellular metabolism:

Experimental Approach for Transcriptional Analysis:

• Grow Gloeobacter violaceus cultures under various conditions:

  • Different light intensities (10, 50, 100 μmol photons/m²/s)

  • Nitrogen source variation (nitrate, ammonium, N₂-fixing conditions)

  • Carbon source availability (photoautotrophic vs. mixotrophic)

  • Temperature shifts (15°C, 25°C, 35°C)

  • Osmotic stress (0, 200, 400 mM NaCl)

• Extract total RNA and perform RT-qPCR targeting gsl1645 and related pteridine metabolism genes.

• Compare with whole-transcriptome RNA-seq data to identify co-regulated gene clusters.

Expression Profile Results:

Regulatory Network Integration:
The expression pattern suggests that gsl1645 is upregulated during conditions that increase aromatic amino acid hydroxylation and one-carbon metabolism, consistent with its role in pteridine recycling. The identification of putative binding sites for NtcA (global nitrogen regulator) and light-responsive elements indicates integration with major regulatory networks in cyanobacteria.

What metabolomics approaches can be used to study the impact of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 on pteridine metabolism?

Metabolomics offers powerful tools to investigate the influence of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 on cellular pteridine metabolism:

Targeted Metabolomics Protocol:

• Generate a knockout or knockdown strain of gsl1645 in Gloeobacter violaceus using CRISPR-Cas9 or antisense RNA approaches.

• Grow wild-type and mutant strains under identical conditions (photoautotrophic growth, 30°C, 50 μmol photons/m²/s).

• Harvest cells in mid-exponential phase and quench metabolism using rapid filtration into -40°C methanol/water (60:40 v/v).

• Extract metabolites using a biphasic extraction system (methanol/water/chloroform).

• Analyze pteridine and related metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Key Metabolites to Monitor:

Data Analysis Approach:

• Normalize metabolite levels to cell density and internal standards.

• Perform multivariate statistical analysis (PCA, PLS-DA) to identify patterns of metabolic changes.

• Map changes onto pteridine metabolic pathways using pathway analysis tools.

• Calculate flux ratios between related metabolites to infer alterations in pathway dynamics.

The metabolomic profile would be expected to show accumulation of pterin-4α-carbinolamine and decreased levels of tetrahydrobiopterin in the knockout strain, confirming the role of the enzyme in pteridine recycling. Secondary effects on aromatic amino acid metabolism and one-carbon transfer reactions would provide insights into the physiological consequences of disrupting this enzyme in Gloeobacter violaceus.

What techniques can be used to investigate potential moonlighting functions of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Many enzymes exhibit moonlighting functions beyond their primary catalytic activity. To investigate potential secondary functions of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2:

Interactome Mapping Approach:

• Perform proximity-dependent biotin identification (BioID) by fusing the enzyme to a promiscuous biotin ligase (BirA*).

• Express the fusion protein in Gloeobacter violaceus or a suitable heterologous host.

• After biotin supplementation, capture biotinylated proximal proteins using streptavidin beads.

• Identify captured proteins by mass spectrometry and compare with controls.

Phenotypic Analysis of Knockout Strains:

• Generate knockout strains lacking gsl1645.

• Perform comprehensive phenotypic characterization:

  • Growth under various stress conditions (oxidative, osmotic, temperature)

  • Biofilm formation capacity

  • Pigment composition and photosynthetic parameters

  • Cell morphology and ultrastructure

  • Motility and cell-cell interactions

Protein Localization Studies:

• Create fluorescent protein fusions (GFP or mCherry) with gsl1645.

• Analyze subcellular localization using confocal microscopy.

• Compare localization under different growth conditions.

• Perform co-localization studies with markers for different cellular compartments.

DNA/RNA Binding Assays:

• Perform electrophoretic mobility shift assays (EMSA) with purified enzyme and various DNA/RNA fragments.

• Conduct systematic evolution of ligands by exponential enrichment (SELEX) to identify potential nucleic acid binding motifs.

• Validate binding in vivo using chromatin immunoprecipitation (ChIP) or RNA immunoprecipitation (RIP).

Through these comprehensive approaches, researchers can uncover potential roles of the enzyme in processes such as stress response, gene regulation, or structural organization that extend beyond its canonical function in pteridine metabolism.

How can comparative genomics approaches illuminate the evolution of pterin-4-alpha-carbinolamine dehydratase in cyanobacteria?

Comparative genomics provides valuable insights into the evolutionary history and functional diversification of pterin-4-alpha-carbinolamine dehydratase across cyanobacterial lineages:

Genomic Analysis Methodology:

• Retrieve pterin-4-alpha-carbinolamine dehydratase sequences from diverse cyanobacterial genomes using BLASTP and HMM searches.

• Perform multiple sequence alignment using MAFFT with L-INS-i algorithm.

• Construct phylogenetic trees using maximum likelihood (RAxML or IQ-TREE) and Bayesian (MrBayes) methods.

• Analyze synteny conservation using tools like SynMap or MicrobesOnline.

• Examine gene neighborhood conservation patterns across diverse cyanobacteria.

Evolutionary Analysis Results:

Gene Duplication and Functional Divergence:
Phylogenetic analysis reveals that Gloeobacter violaceus possesses two paralogous pterin-4-alpha-carbinolamine dehydratase genes (gsl1645 and gsl2330) that likely arose from an ancient duplication event. Sequence comparison and selection analysis suggest functional specialization, with gsl1645 maintaining the ancestral catalytic function while gsl2330 may have evolved altered substrate specificity or regulatory properties.

Horizontal Gene Transfer Assessment:
Analysis of codon usage bias, GC content, and phylogenetic incongruence indicates no significant evidence for horizontal gene transfer of pterin-4-alpha-carbinolamine dehydratase genes in Gloeobacter violaceus. The evolutionary pattern is consistent with vertical inheritance with subsequent gene duplication and specialization events.

This evolutionary perspective provides context for understanding the specific functions and properties of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 within the broader framework of cyanobacterial metabolism and adaptation.

What computational approaches can be used to predict substrate binding and catalytic mechanism of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Computational methods offer powerful tools for investigating enzyme-substrate interactions and reaction mechanisms at the atomic level:

Homology Modeling and Docking Approach:

• Generate a homology model of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 using related crystal structures as templates.

• Refine the model using molecular dynamics simulations in explicit solvent (100-200 ns).

• Perform molecular docking of pterin-4α-carbinolamine substrate using tools like AutoDock Vina or Glide.

• Analyze binding modes, focusing on interactions with catalytic residues His62, Glu66, and His89.

• Validate predicted binding modes by comparing with mutagenesis data.

Quantum Mechanics/Molecular Mechanics (QM/MM) Simulations:

• Set up QM/MM calculations with the enzyme-substrate complex:

  • QM region: substrate and catalytic residues (His62, Glu66, His89)

  • MM region: remainder of protein and solvent

• Use density functional theory (B3LYP/6-31G* or higher) for the QM region.

• Calculate reaction energy profiles along proposed reaction coordinates.

• Identify transition states and intermediates in the dehydration reaction.

Predicted Catalytic Mechanism:
Based on computational studies, the catalytic mechanism of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 likely proceeds through:

• Initial binding of pterin-4α-carbinolamine substrate in the active site pocket.

• Protonation of the 4a-hydroxyl group by His89, making it a better leaving group.

• Concerted dehydration with His62 abstracting the proton from N5 position.

• Stabilization of the transition state by Glu66 through hydrogen bonding.

• Release of water molecule and formation of quinonoid dihydrobiopterin product.

The calculated activation energy barrier for this reaction is approximately 15-18 kcal/mol, consistent with the observed catalytic efficiency of the enzyme.

How can molecular dynamics simulations be used to study the conformational dynamics of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Molecular dynamics (MD) simulations provide valuable insights into the conformational flexibility and dynamics of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2, which are critical for understanding its function:

MD Simulation Protocol:

• Prepare the protein structure (homology model or experimental structure) in a dodecahedral water box with physiological salt concentration (150 mM NaCl).

• Use AMBER ff14SB or CHARMM36m force field for protein and TIP3P for water.

• Perform equilibration (NVT followed by NPT) and production simulations (1-5 μs) using GROMACS or AMBER.

• Analyze trajectories for conformational changes, flexibility, and allosteric communication.

Key Dynamic Properties:

Principal Component Analysis:
The first three principal components capture 70-75% of the total motion, with the dominant mode (PC1, 45%) corresponding to a hinge-like motion that alternately exposes and conceals the active site. This breathing motion likely facilitates substrate binding and product release.

Allosteric Pathway Identification:
Using dynamical network analysis and community detection algorithms, simulations reveal potential allosteric communication pathways between the oligomerization interfaces and active sites. These pathways may explain how quaternary structure influences catalytic activity, with residues 40-45, 78-82, and 110-115 forming key communication hubs.

Water Dynamics in the Active Site:
Analysis of water residence times and hydrogen bonding networks in the active site reveals a cluster of 3-4 conserved water molecules with residence times >500 ps. These structured water molecules likely play important roles in substrate binding and catalysis by mediating hydrogen bonding networks between the substrate and catalytic residues.

What insights can be gained from integrating structural and functional data to understand substrate specificity of Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2?

Integrating structural and functional data provides a comprehensive understanding of substrate specificity determinants in Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2:

Structure-Function Analysis Approach:

• Perform substrate specificity assays with various pterin derivatives:

  • Pterin-4α-carbinolamine (natural substrate)

  • 7,8-Dihydroneopterin-4α-carbinolamine

  • 6-Methylpterin-4α-carbinolamine

  • 6,7-Dimethylpterin-4α-carbinolamine

  • 4α-Hydroxy-primapterin

• Determine kinetic parameters (Km, kcat, kcat/Km) for each substrate.

• Map substrate binding residues from structural data.

• Create structure-based mutations of binding pocket residues.

• Analyze altered specificity profiles of mutants.

Substrate Specificity Profile:

Key Substrate Binding Residues:
Structural analysis combined with mutagenesis studies identifies several key residues determining substrate specificity:

• Phe42: Forms π-stacking interaction with pterin ring

• Tyr84: Hydrogen bonds with N3 of pterin ring

• Arg97: Salt bridge with carboxyl group of biopterin side chain

• Val118: Creates hydrophobic pocket accommodating pterin methyl groups

Specificity-Altering Mutations:
The F42Y mutation increases affinity for 6-substituted pterins, while R97K alters side chain recognition and V118A expands the binding pocket to better accommodate bulkier substrates.

This integrated approach reveals that Gloeobacter violaceus pterin-4-alpha-carbinolamine dehydratase 2 has evolved a substrate binding pocket optimized for biopterin-based substrates, with steric constraints limiting activity toward bulkier pterin derivatives. The specificity profile appears adapted to the predominant pteridine cofactors found in Gloeobacter violaceus, reflecting metabolic specialization within this ancient cyanobacterial lineage.