Recombinant Prochlorococcus marinus subsp. pastoris 3-dehydroquinate dehydratase (aroQ)

What is the role of 3-dehydroquinate dehydratase (aroQ) in Prochlorococcus marinus metabolism?

3-Dehydroquinate dehydratase (DHQD, aroQ) catalyzes the third step in the shikimate pathway, converting 3-dehydroquinic acid (DHQ) to 3-dehydroshikimic acid (DHS). This enzymatic reaction is crucial for the biosynthesis of aromatic amino acids and folates, which are essential metabolites for bacterial survival. In P. marinus, this pathway provides precursors for the synthesis of chorismate, which subsequently leads to the biosynthesis of phenylalanine, tyrosine, and tryptophan .

The reaction catalyzed is specifically a dehydration, eliminating water from DHQ to form DHS, which serves as a critical intermediate in the multi-step shikimate pathway. The enzyme belongs to type II DHQDs, which catalyze anti-dehydration through an enolate intermediate, forming a Schiff base with a conserved lysine residue in the active site .

How is the aroQ gene organized in the genome of Prochlorococcus marinus subsp. pastoris?

Prochlorococcus marinus subsp. pastoris, also known as strain CCMP1986 or MED4, has one of the smallest genomes of any photosynthetic organism, consisting of a single circular chromosome of only 1,657,990 bp containing 1,796 predicted protein-coding genes . Within this compact genome, the aroQ gene exists as part of the cyanobacterium's essential metabolic machinery.

Unlike many other bacterial species where aroQ frequently occurs as a fusion with other aromatic pathway genes (such as pheA or tyrA), in P. marinus it appears to exist as a standalone gene. This differs from organisms like Erwinia herbicola where aroQ has been found to have a cleavable signal peptide and is located in the periplasmic compartment .

The aroQ gene in P. marinus is functionally connected to other aromatic pathway genes in its metabolic network, as demonstrated by protein-protein interaction data:

This network of interactions highlights aroQ's integration into the larger shikimate pathway machinery .

What type of 3-dehydroquinate dehydratase is encoded by the aroQ gene in Prochlorococcus marinus?

The aroQ gene in Prochlorococcus marinus encodes a Type II dehydroquinate dehydratase (DHQD). DHQDs are classified into two distinct types based on their structure, oligomeric state, and catalytic mechanism:

• Type I DHQD (aroD): Catalyzes syn-dehydration through a covalent imine intermediate. These enzymes have an (α/β)8 fold and exist as homodimers .

• Type II DHQD (aroQ): Catalyzes anti-dehydration by forming a Schiff base with a conserved lysine residue through an enolate intermediate. These enzymes exist as homododecamers containing a flavodoxin fold .

The Type II DHQDs, including the one from P. marinus, form complex quaternary structures consisting of four trimers with three interfacial active sites . This structural arrangement is important for the enzyme's function and stability.

What is the structural classification of AroQ proteins and how does the P. marinus enzyme compare?

AroQ proteins (Type II DHQDs) belong to a structurally distinct class that differs significantly from Type I DHQDs. Based on structural studies of similar enzymes, AroQ proteins typically:

• Form homododecameric structures consisting of four trimers

• Contain a flavodoxin fold in each monomer

• Have three interfacial active sites per trimer

• Possess a distinctive lid loop that can adopt open and closed conformations

A periplasmic subclass of AroQ has been identified in some bacteria, such as Salmonella typhimurium and Pseudomonas aeruginosa, which are approximately twice the size of cytoplasmic AroQ proteins due to a carboxy-terminal extension of unknown function .

While the specific structure of P. marinus AroQ has not been directly reported in the search results, structural comparison with homologs from other bacteria can provide insight into its likely structural features. The quaternary structure plays a crucial role in the enzyme's stability and function, with interactions between subunits contributing to the formation of catalytically active interfaces.

How does the active site of 3-dehydroquinate dehydratase from P. marinus function during catalysis?

Type II DHQDs, including the AroQ from P. marinus, catalyze the conversion of DHQ to DHS through an anti-elimination mechanism. The catalytic process involves:

• Binding of the substrate (DHQ) in the active site

• Formation of a Schiff base between the substrate and a conserved lysine residue

• Generation of an enolate intermediate

• Anti-elimination of water to form DHS

Studies on similar Type II DHQDs have identified key residues involved in catalysis. For example, in Corynebacterium glutamicum DHQD (CgDHQD), residues such as R19, S103, and P105 play important roles in substrate binding and catalysis . Specifically:

• R19 likely interacts with the carboxyl group of DHQ

• S103 is positioned near the 5-hydroxyl group of DHQ

• P105 is a distinctive residue in some Corynebacterium species that affects enzyme activity

Replacement of P105 with isoleucine or valine (conserved in other DHQDs) caused approximately 70% decrease in activity, while replacement of S103 with threonine increased activity by 10% . These structure-function relationships provide valuable insights into the catalytic mechanism of Type II DHQDs.

Are there any known inhibitors of Prochlorococcus marinus aroQ, and how do they affect enzyme activity?

While specific inhibitors of P. marinus aroQ have not been directly reported in the search results, studies on similar Type II DHQDs provide valuable insights into potential inhibition mechanisms:

• Citrate inhibition: In CgDHQD, citrate has been identified as an inhibitor that binds to the active site with a half-opened lid loop . The crystal structure of CgDHQD with bound citrate was determined at a resolution of 1.80 Å, revealing details of this inhibitor-enzyme interaction.

• Competitive inhibitors: Compounds structurally similar to the substrate (DHQ) or product (DHS) may act as competitive inhibitors by occupying the active site.

• Transition state analogs: Compounds that mimic the transition state of the reaction are potential potent inhibitors of the enzyme.

Understanding inhibition mechanisms is important for:

• Developing tools to study enzyme function

• Potential antimicrobial drug development, as the shikimate pathway is absent in mammals

• Elucidating the structural basis of substrate specificity

For researchers investigating P. marinus aroQ, testing known inhibitors of related DHQDs would be a valuable approach to characterizing its inhibition profile.

What expression systems are most effective for producing recombinant P. marinus aroQ protein?

Based on available information for similar recombinant proteins from P. marinus and other organisms, several expression systems have been successfully employed:

• Escherichia coli-based expression: The most commonly used system for recombinant protein production. For similar enzymes like CgDHQD, expression in E. coli BL21(DE3) T1R strain using a pET30a vector with a C-terminal 6x-His tag has proven successful . Protein expression can be induced with IPTG (typically 0.5 mM) at reduced temperatures (e.g., 291 K for 20 hours) to enhance protein solubility.

• Yeast expression systems: According to the search results, some commercial recombinant proteins from P. marinus are produced in yeast expression systems . Yeast can provide advantages for proteins requiring eukaryotic post-translational modifications, although this is generally less critical for bacterial proteins.

For optimal expression of recombinant P. marinus aroQ, researchers should consider:

• Codon optimization for the expression host

• Selection of appropriate promoters (e.g., T7 for E. coli)

• Inclusion of purification tags (His-tag, GST-tag, etc.)

• Optimization of induction conditions (temperature, inducer concentration, duration)

• Use of specialty strains designed to express proteins that may be toxic or form inclusion bodies

What purification strategy should be employed to obtain high-purity recombinant P. marinus aroQ?

A multi-step purification strategy is recommended to obtain high-purity recombinant P. marinus aroQ, based on successful approaches used for similar enzymes:

• Initial capture: Affinity chromatography using Ni-NTA agarose for His-tagged proteins. After cell lysis by ultrasonication and clarification by centrifugation, the soluble fraction is applied to the Ni-NTA column. Washing with buffer containing low imidazole concentrations (e.g., 20 mM) removes weakly bound proteins, followed by elution with higher imidazole concentrations (e.g., 300 mM) .

• Intermediate purification: Ion-exchange chromatography (e.g., HiTrap Q FF) can separate proteins based on charge differences, removing contaminants with different isoelectric points .

• Polishing step: Size exclusion chromatography (e.g., HiPrep 26/60 Sephacryl S-300 HR column) for final purification, which separates proteins based on molecular size and shape .

• Quality control: The purified protein should be assessed by:

  • SDS-PAGE to verify purity and molecular weight

  • Western blotting if specific antibodies are available

  • Activity assays to confirm enzymatic function

  • Mass spectrometry to verify protein identity

Using this approach, researchers have achieved high-purity preparations of similar enzymes. For example, CgDHQD was purified to a state where a single band was visible by SDS-PAGE after Coomassie blue staining .

How stable is recombinant P. marinus aroQ during storage, and what conditions maximize its shelf life?

Based on information from commercial recombinant proteins and similar enzymes, the following recommendations can be made for maximizing the stability and shelf life of recombinant P. marinus aroQ:

Storage conditions and shelf life:

• Liquid form: Approximately 6 months at -20°C/-80°C

• Lyophilized form: Approximately 12 months at -20°C/-80°C

• Working aliquots: Up to one week at 4°C

Recommended storage buffer components:

• Buffer: Typically Tris-based (40-50 mM, pH 7.5-8.0)

• Cryoprotectant: 20-50% glycerol for frozen storage

• Optional additives: Small amounts of reducing agents (e.g., DTT, β-mercaptoethanol) may help maintain activity

Reconstitution guidelines:

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

• Addition of glycerol to a final concentration of 5-50% is recommended for long-term storage

• Centrifugation of the vial before opening helps bring contents to the bottom

Important precautions:

• Avoid repeated freeze-thaw cycles

• Aliquot the protein solution into single-use volumes before freezing

• Store working stocks at 4°C only for short periods (≤1 week)

• Avoid exposure to extreme pH, high temperatures, or strong denaturants

By following these guidelines, researchers can maximize the stability and activity of recombinant P. marinus aroQ during storage and experimental use.

How does the aroQ gene from Prochlorococcus marinus compare to similar genes in other cyanobacteria?

Prochlorococcus marinus has unique adaptations compared to typical cyanobacteria, and these differences extend to its metabolic pathways. Regarding the aroQ gene:

• Genomic context: In P. marinus, aroQ exists within a highly compact genome, reflecting the organism's evolutionary trajectory toward genome minimization . This contrasts with many other cyanobacteria that have larger genomes.

• Light adaptation influence: P. marinus strains are adapted to either high-light or low-light conditions, which affects their genomic content. The high-light-adapted strains like P. marinus subsp. pastoris (MED4) have undergone more extensive genome reduction compared to low-light-adapted strains .

• Protein interaction network: In P. marinus, aroQ interactions with other shikimate pathway enzymes (aroB, aroA, aroE, aroC) as well as with aromatic amino acid biosynthesis enzymes (tyrA, pheA) have been identified through computational approaches . This interaction network highlights the integration of aroQ in the metabolic framework specific to this organism.

• Evolutionary conservation: While the shikimate pathway is generally conserved across bacteria, specific adaptations in enzyme sequence, regulation, and cellular localization can differ. In some bacteria, periplasmic forms of aroQ have been identified, whereas in others, the enzyme is cytoplasmic or exists as a fusion protein with other enzymatic domains .

The evolution of aroQ in P. marinus likely reflects the environmental pressures faced by this organism in nutrient-limited oceanic environments, where efficient metabolism with minimal genetic content provides a selective advantage.

What factors contributed to the evolution of different types of dehydroquinate dehydratases across bacterial species?

The evolution of two distinct types of dehydroquinate dehydratases (Type I/aroD and Type II/aroQ) across bacterial species reflects diverse selective pressures and evolutionary processes:

• Structural and mechanistic divergence: Type I and Type II DHQDs catalyze the same reaction but through different mechanisms:

  • Type I enzymes catalyze syn-dehydration via a covalent imine intermediate

  • Type II enzymes catalyze anti-dehydration through an enolate intermediate

• Domain fusions and protein evolution: AroQ has been an extremely popular partner for fusion with other aromatic pathway genes, highlighting evolutionary forces driving the co-localization of functionally related enzymes . Examples include:

  • aroQ- pheA fusion in gamma and beta proteobacteria

  • aroQ- tyrA fusion in enteric bacteria

  • aroQ- aroD fusion in Clostridium acetobutylicum

  • aroQ- aroA fusion in Bacillus subtilis, Staphylococcus, and Deinococcus

• Functional advantages: In B. subtilis, the AroQ domain functions not only as a catalytic domain for chorismate mutase but also as the allosteric domain for the fused AroA domain, providing a physical basis for sequential feedback inhibition .

• Cellular localization adaptations: The emergence of periplasmic AroQ proteins with signal peptides and C-terminal extensions represents another evolutionary trajectory, potentially related to metabolic compartmentalization .

These evolutionary patterns suggest that DHQDs have undergone significant diversification in response to metabolic requirements, regulatory pressures, and cellular organization across different bacterial lineages.

Is there evidence for horizontal gene transfer of aroQ in Prochlorococcus marinus lineages?

While direct evidence for horizontal gene transfer (HGT) of aroQ in Prochlorococcus marinus is not explicitly stated in the search results, several factors suggest this possibility:

• Distribution of periplasmic aroQ: The erratic phylogenetic distribution of periplasmic AroQ (designated as *AroQ) may be explained by various mechanisms, including "massive gene loss, horizontal transfer or independent evolution of a signal peptide and carboxy-terminal extension" . This suggests HGT as one potential mechanism for the spread of this gene variant across unrelated bacteria.

• Genomic diversity in Prochlorococcus: Despite having small individual genomes, Prochlorococcus strains collectively show remarkable genetic diversity. The pangenome of Prochlorococcus contains more than 80,000 genes , indicating substantial genetic exchange within this genus.

• Ecosystem context: Prochlorococcus exists in microbial communities with diverse bacteria and phages, providing opportunities for genetic exchange through various HGT mechanisms.

• Gene content variation: Differences in gene content between high-light and low-light adapted Prochlorococcus strains could potentially be influenced by HGT events in addition to gene loss processes.

To definitively determine if aroQ in P. marinus resulted from HGT would require comprehensive phylogenetic analyses comparing:

• Gene phylogeny versus species phylogeny

• GC content and codon usage patterns compared to the genomic average

• Presence of nearby mobile genetic elements or HGT signatures

• Distribution patterns across related and unrelated bacterial species

Such analyses could reveal whether aroQ in P. marinus represents an ancestral gene or one acquired through horizontal transfer.

What are the optimal conditions for measuring the enzymatic activity of recombinant P. marinus aroQ?

Based on methods used for similar dehydroquinate dehydratases, the following protocol can be optimized for measuring the enzymatic activity of recombinant P. marinus aroQ:

Spectrophotometric assay conditions:

• Reaction buffer: 50 mM Tris-HCl, pH 8.0

• Substrate: 3-dehydroquinic acid potassium salt (CAS No. 494211-79-9), with concentrations ranging from 50 to 2,000 μM for kinetic studies

• Enzyme concentration: Approximately 20 nM (may require optimization)

• Detection method: Monitor the increased absorbance of 3-dehydroshikimic acid (DHS) at 234 nm (ε = 1.2 × 10^4 M^-1cm^-1) using an ultraviolet spectrophotometer

• Temperature: Typically room temperature (25°C) or physiologically relevant temperature

• Reaction initiation: Pre-incubate buffer and substrate for one minute, then add enzyme to initiate the reaction

Considerations for optimal assay performance:

• Enzyme stability: Ensure the enzyme remains stable throughout the measurement period

• Linear range: Determine the linear range of the assay with respect to time and enzyme concentration

• Controls: Include appropriate controls:

  • No-enzyme control to account for non-enzymatic conversion

  • Heat-inactivated enzyme control

  • Known inhibitor control (e.g., citrate) to confirm specific activity

• Data collection: Collect multiple time points to calculate initial velocity accurately

Alternative assay methods:

• HPLC: For more sensitive detection or confirmation of product identity

• Coupled enzyme assays: If direct spectrophotometric measurement is challenging

• Mass spectrometry: For detailed product analysis or reaction mechanism studies

By optimizing these conditions, researchers can obtain reliable measurements of P. marinus aroQ enzymatic activity for kinetic characterization and inhibitor studies.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of P. marinus aroQ?

Site-directed mutagenesis is a powerful approach for investigating enzyme catalytic mechanisms by systematically altering key residues. For P. marinus aroQ, the following methodology can be implemented:

Experimental approach:

• Identification of target residues: Based on sequence alignments with well-characterized DHQDs, homology modeling, and/or crystal structures, identify conserved residues likely involved in:

  • Substrate binding

  • Catalysis

  • Maintenance of active site architecture

  • Conformational changes during catalysis

• Mutagenesis method: Use a QuikChange site-directed mutagenesis kit or similar approach to introduce specific mutations . This typically involves:

  • Designing complementary primers containing the desired mutation

  • PCR amplification of the entire plasmid

  • DpnI digestion to remove template DNA

  • Transformation into competent E. coli cells

• Mutation strategies:

  • Conservative substitutions: Replace residues with chemically similar amino acids (e.g., Lys→Arg, Asp→Glu)

  • Non-conservative substitutions: Replace with functionally distinct amino acids

  • Alanine scanning: Systematically replace residues with alanine to assess their contribution

• Validation: Confirm mutations by DNA sequencing before proceeding to protein expression and purification

Analysis of mutant enzymes:

• Kinetic characterization: Determine Km, kcat, and kcat/Km values for each mutant compared to wild-type

• Substrate specificity: Test alternative substrates to assess changes in specificity

• pH-activity profiles: Determine if mutations alter the pH optimum or dependence

• Inhibitor sensitivity: Test sensitivity to known inhibitors

• Structural studies: When possible, determine crystal structures of key mutants

Example mutations based on similar DHQDs:
Findings from CgDHQD suggest the following mutations might be informative:

• Conserved arginine residues involved in substrate binding

• Serine residues near the 5-hydroxyl group of DHQ

• Unique proline residues that may affect activity

• Lysine residues involved in Schiff base formation

By systematically analyzing the effects of these mutations on enzyme activity and structure, researchers can elucidate the catalytic mechanism of P. marinus aroQ and identify key residues essential for function.

What techniques are available for studying the quaternary structure of recombinant P. marinus aroQ?

Understanding the quaternary structure of P. marinus aroQ is crucial for elucidating its function, as Type II DHQDs typically form complex oligomeric assemblies. Several complementary techniques can be employed:

Structural biology techniques:

• X-ray crystallography: The gold standard for high-resolution structural determination. Crystal structures of similar DHQDs have been determined at resolutions of 1.8-2.0 Å, revealing detailed quaternary arrangements . Key steps include:

  • Crystallization screening using sparse-matrix approaches

  • Optimization of crystallization conditions

  • Data collection at synchrotron sources

  • Phase determination and structure refinement

• Cryo-electron microscopy (cryo-EM): Particularly useful for large protein complexes, allowing visualization of quaternary structure without crystallization.

• Chemical crosslinking combined with mass spectrometry: Identifies residues in close proximity, providing information about subunit interfaces.

Biophysical characterization:

• Differential scanning calorimetry (DSC): Measures thermal stability, which can differ between monomeric and oligomeric forms.

• Circular dichroism (CD): Assesses secondary structure content and thermal stability.

• Native PAGE: Provides information about the native oligomeric state and homogeneity.

For comprehensive characterization of P. marinus aroQ quaternary structure, a combination of these techniques would provide complementary information about its oligomeric state, assembly mechanism, and structural stability under various conditions.

How should kinetic data for recombinant P. marinus aroQ be analyzed to determine Michaelis-Menten parameters?

Proper analysis of enzyme kinetic data for P. marinus aroQ requires rigorous methodological approaches to obtain accurate Michaelis-Menten parameters:

Experimental design for kinetic measurements:

• Substrate range: Use 8-12 substrate concentrations ranging from 0.2×Km to 5×Km (typically 50-2,000 μM for DHQDs)

• Enzyme concentration: Use the minimum concentration that gives reliable rate measurements (approximately 20 nM for similar DHQDs)

• Initial velocity conditions: Ensure measurements are taken during the linear phase of the reaction (<10% substrate conversion)

• Replication: Perform at least triplicate measurements at each substrate concentration

Data analysis workflow:

• Initial velocity calculation: Determine the rate (v) at each substrate concentration [S] from the linear portion of progress curves

• Primary data visualization: Plot reaction velocity (v) versus substrate concentration [S] to visualize the hyperbolic relationship

• Non-linear regression analysis: Fit data directly to the Michaelis-Menten equation:
  v = Vmax × [S] / (Km + [S])
  using non-linear regression software (e.g., GraphPad Prism, Origin, R)

• Linear transformations for verification and visualization:

  • Lineweaver-Burk plot (1/v vs. 1/[S])

  • Eadie-Hofstee plot (v vs. v/[S])

  • Hanes-Woolf plot ([S]/v vs. [S])

• Parameter extraction: Determine:

  • Km (Michaelis constant): Substrate concentration at half-maximal velocity

  • Vmax (maximum velocity): Asymptotic maximum rate at saturating substrate

  • kcat (turnover number): Calculate as Vmax/[E]total

  • kcat/Km (catalytic efficiency): Often the most relevant parameter for comparing enzyme variants

• Statistical analysis: Report 95% confidence intervals for all parameters and evaluate goodness of fit (R² values)

Handling special cases:

• Substrate inhibition: If velocity decreases at high [S], fit to the modified equation:
  v = Vmax × [S] / (Km + [S] + [S]²/Ki)

• Sigmoidal kinetics: If the enzyme shows cooperativity, use the Hill equation:
  v = Vmax × [S]^h / (K'+ [S]^h)

• Non-ideal behavior: Address potential issues like substrate depletion, product inhibition, or enzyme instability by appropriate controls and corrections

By following these methodological approaches, researchers can obtain reliable kinetic parameters for P. marinus aroQ that facilitate comparison with other DHQDs and assessment of mutagenesis effects.

What computational approaches can be used to predict substrate specificity of P. marinus aroQ based on its sequence?

Multiple computational methods can be integrated to predict substrate specificity of P. marinus aroQ from its amino acid sequence:

Sequence-based approaches:

• Multiple sequence alignment (MSA): Align the P. marinus aroQ sequence with well-characterized DHQDs to identify conserved residues in the active site. Tools like Clustal Omega, MUSCLE, or T-Coffee can be used.

• Motif identification: Search for conserved motifs associated with substrate binding using tools like MEME, PROSITE, or InterProScan.

• Phylogenetic analysis: Construct phylogenetic trees to identify the closest characterized homologs, which may share similar specificity profiles. This helps place P. marinus aroQ within the context of known DHQD subfamilies.

• Conservation analysis: Use tools like ConSurf to map conservation scores onto protein structures, highlighting functionally important residues.

Structure-based predictions:

• Homology modeling: Generate a 3D structural model of P. marinus aroQ using templates from closely related DHQDs with known structures. Programs like SWISS-MODEL, Phyre2, or I-TASSER are suitable for this purpose.

• Active site analysis: Identify the binding pocket and catalytic residues using CASTp, SiteMap, or similar tools. Compare with known DHQD structures to identify similarities and differences that might affect specificity.

• Molecular docking: Dock potential substrates into the predicted active site using software like AutoDock, GOLD, or Glide to assess binding poses and energetics.

• Molecular dynamics simulations: Perform MD simulations of the enzyme-substrate complex to assess stability and dynamics of binding interactions.

Integrated computational approaches:

• Quantitative structure-activity relationship (QSAR): Develop models relating enzyme sequence features to substrate specificity patterns.

• Machine learning methods: Use supervised learning approaches trained on known DHQD data to predict specificity of novel enzymes.

• Network analysis: Examine the genomic context and metabolic network to predict likely substrates based on pathway relationships.

Case example from research:
Structural comparison of CgDHQD with a homolog from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site that affect substrate binding . Particularly, CgDHQD possesses a distinctive proline residue (P105) not conserved in other DHQDs. Replacement of this residue significantly decreased enzyme activity, highlighting how computational identification of unique residues can guide experimental work .

By integrating these computational approaches, researchers can generate testable hypotheses about P. marinus aroQ substrate specificity and guide experimental design for biochemical characterization.

How can structural data from crystallography be integrated with biochemical findings to understand the function of P. marinus aroQ?

Integrating crystallographic structural data with biochemical findings provides comprehensive insights into P. marinus aroQ function through a multidisciplinary approach:

Integration workflow:

• Structure determination and analysis:

  • Solve the crystal structure of P. marinus aroQ at high resolution

  • Identify key structural features: active site architecture, quaternary structure, mobile elements

  • When multiple structures are available (e.g., with/without ligands), analyze conformational changes

• Structure-guided biochemical investigation:

  • Design site-directed mutagenesis experiments targeting residues identified in the structure

  • Perform enzyme kinetics on wild-type and mutant proteins

  • Investigate substrate specificity and inhibitor binding

  • Analyze the effects of pH, temperature, and buffer conditions on structure stability

• Iterative refinement:

  • Use biochemical data to inform additional structural studies

  • Crystallize enzyme-substrate or enzyme-inhibitor complexes based on biochemical findings

  • Target mobile regions or alternative conformations identified through biochemistry

Example integration from similar DHQDs:
In studies of CgDHQD, researchers determined two crystal structures :

• Wild-type CgDHQD with citrate (inhibitor) at 1.80 Å resolution

• CgDHQD R19A mutant with DHQ (substrate) complexed at 2.00 Å resolution

These structures revealed:

• The DHQ-binding site and interaction patterns

• An unusual binding mode of citrate inhibitor with a half-opened lid loop

• Structural variations compared to homologs from other species

Based on these structural insights, biochemical experiments were designed:

• Mutations of specific residues (P105, S103) identified from structural comparisons

• Activity assays showing ~70% decrease in activity when P105 was replaced with isoleucine or valine

• 10% increase in activity when S103 was replaced with threonine

The crystallography data provided the following parameters, which were essential for interpreting biochemical results:

This integration of structural and biochemical data provided a comprehensive understanding of how specific residues contribute to enzyme function, explaining variation in reaction efficiency due to structural differences .