Recombinant Methanococcus maripaludis 3-dehydroquinate synthase (MMP0006)

What is the biochemical function of 3-dehydroquinate synthase in M. maripaludis?

3-dehydroquinate synthase (DHQS, EC 4.2.3.4) catalyzes the second step in the shikimate pathway, converting 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to 3-dehydroquinate (DHQ). In M. maripaludis, this enzyme is part of the de novo aromatic amino acid biosynthesis pathway that produces chorismate as an intermediate. The reaction involves several chemical steps including alcohol oxidation, β-elimination of phosphate, carbonyl reduction, and intramolecular aldol condensation, all occurring at a single active site.

The significance of this enzyme is highlighted by studies showing that deletion of 3-dehydroquinate dehydratase (DHQ), which catalyzes the step immediately following DHQS, results in aromatic amino acid auxotrophy in M. maripaludis . This indicates that the de novo pathway is essential when exogenous aryl acids are not available. DHQS represents a potential target for archaeal metabolism research and antimicrobial development, as the shikimate pathway is absent in mammals.

How does M. maripaludis utilize multiple pathways for aromatic amino acid biosynthesis?

M. maripaludis possesses remarkable metabolic flexibility for aromatic amino acid biosynthesis through three distinct pathways:

• The canonical de novo pathway: This pathway involves DHQS (MMP0006) and proceeds through chorismate to produce all three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) . This pathway is essential when alternative sources are unavailable.

• The aryl acid incorporation pathway: M. maripaludis can incorporate exogenous aryl acids (phenylacetate, indoleacetate, and p-hydroxyphenylacetate) via indolepyruvate oxidoreductase (IOR) to produce aromatic amino acids, bypassing the need for de novo synthesis .

• The novel DKFP pathway: Recent research has identified a unique 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for aromatic amino acid biosynthesis that does not involve chorismate as an intermediate .

This metabolic versatility allows M. maripaludis to adapt to different environmental conditions. When aryl acids are available, the activity of the DKFP pathway is reduced, indicating regulatory cross-talk between these pathways . This multiple pathway strategy may be particularly advantageous for an organism that lives in anaerobic sediments where nutrient availability can fluctuate.

What expression systems are optimal for producing recombinant M. maripaludis DHQS?

Based on successful expression of other proteins from M. maripaludis and related archaea, several expression systems should be considered for recombinant DHQS production:

• E. coli expression systems: The pET vector system in E. coli BL21(DE3) is a primary choice for archaeal enzyme expression . This system offers high-level protein production using T7 promoter-driven expression with IPTG induction. The search results indicate successful use of E. coli BL21(DE3)/pET28a for recombinant protein expression, with optimizable parameters including rotary speed, induction temperature, and induction time .

• Temperature considerations: Lower induction temperatures (17°C) have been shown to significantly improve solubility and activity of recombinant proteins in E. coli . This is particularly important for archaeal proteins that may face folding challenges in bacterial hosts.

• Potential challenges and solutions:

  • Codon optimization for E. coli expression

  • Addition of solubility tags (His, MBP, SUMO)

  • Co-expression with chaperones to improve folding

  • Consideration of homologous expression in M. maripaludis itself for proteins that prove difficult to express heterologously

What purification strategies yield active recombinant DHQS from M. maripaludis?

A multi-step purification strategy is recommended for obtaining pure, active recombinant DHQS:

• Initial capture: Affinity chromatography using His-tag (if expressed with pET vectors) provides a convenient first step. Immobilized metal affinity chromatography (IMAC) with Ni-NTA resin is effective for capturing His-tagged proteins with minimal non-specific binding.

• Secondary purification: Ion exchange chromatography based on the predicted isoelectric point of DHQS can remove remaining contaminants. The buffer system should maintain enzyme stability with appropriate pH, salt concentration, and potential additives.

• Polishing: Size exclusion chromatography separates any aggregates or oligomeric forms and ensures high purity for enzymatic and structural studies.

• Buffer considerations: Throughout purification, buffers should contain components that maintain DHQS stability and activity:

  • Divalent metal ions (Co^2+ may be particularly important, as other M. maripaludis enzymes show strong Co^2+ preference)

  • NAD+ as a cofactor for DHQS

  • Reducing agents to prevent oxidation of cysteine residues

  • Glycerol or other stabilizing agents

• Activity validation: After purification, enzyme activity should be verified using the coupled enzyme assay that measures the formation of 3-dehydroshikimate spectrophotometrically at 234 nm (ε = 12 × 10^3 M^-1 cm^-1) . This involves the DHQS-catalyzed conversion of DAH7P to DHQ followed by the 3-dehydroquinase-catalyzed conversion of DHQ to 3-dehydroshikimate.

How does plasmid stability affect recombinant DHQS expression in E. coli?

Plasmid stability significantly impacts recombinant protein yield and quality in E. coli expression systems. Research indicates several important considerations for maintaining plasmid stability during DHQS expression:

• Selection pressure: Continuous antibiotic selection is crucial for maintaining plasmids in expression cultures. Studies with recombinant E. coli showed that plasmid stability can be assessed by comparing colony growth on selective versus non-selective media .

• Copy number optimization: Plasmid copy number directly affects protein yield. Research demonstrates that simulated microgravity (SMG) conditions can increase plasmid copy number in E. coli, potentially enhancing recombinant protein production . After induction at various temperatures (17, 27, and 37°C), plasmid DNA concentration can be quantified using spectrophotometry to calculate plasmid number per cell .

• Growth conditions: Environmental factors significantly influence plasmid stability and copy number. Optimal conditions found for recombinant protein expression in E. coli include:

  • Induction temperature: 17°C

  • Rotary speed: 15 rpm

  • Induction time: 4 hours

  • IPTG concentration: 0.8 mM

• Metabolic burden: High-level expression of recombinant proteins creates metabolic stress that can lead to plasmid loss. Transcriptomic analysis of recombinant E. coli revealed upregulation of energy metabolism genes that may help maintain plasmid stability during protein production .

What assay methods can quantify DHQS activity and determine kinetic parameters?

The gold standard for measuring DHQS activity is a coupled enzyme continuous assay that provides quantitative kinetic data:

• Assay principle: The assay monitors the sequential conversion of DAH7P to DHQ (catalyzed by DHQS) and then to 3-dehydroshikimate (catalyzed by added 3-dehydroquinase) . The formation of 3-dehydroshikimate is measured spectrophotometrically at 234 nm using its extinction coefficient (ε = 1.2 × 10^4 M^-1 cm^-1) .

• Assay conditions:

  • Buffer: Potassium-PIPES buffer (50 mM, pH 7.0)

  • Substrate: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P)

  • Temperature: 37°C for M. maripaludis DHQS (mesophilic archaea) , compared to 60°C for Pyrococcus DHQS (hyperthermophilic archaea)

  • Excess 3-dehydroquinase: At least 10 times the concentration of DHQS to ensure the first step is rate-limiting

• Substrate preparation: DAH7P can be enzymatically synthesized using DAH7P synthase or chemically synthesized according to published protocols.

• Analysis of kinetic parameters:

  • Km for DAH7P: Vary substrate concentration while maintaining constant cofactor levels

  • Km for NAD+: Vary cofactor concentration with saturating substrate

  • kcat: Calculate from Vmax and enzyme concentration

  • Effects of potential inhibitors: Add candidate compounds and determine inhibition constants

• Alternative assays: For high-throughput screening or specific applications, endpoint assays measuring DHQ formation by colorimetric methods or LC-MS can be employed.

How do metal ions and cofactors influence DHQS activity from M. maripaludis?

DHQS requires both NAD+ as a cofactor and divalent metal ions for catalytic activity. While specific data for M. maripaludis DHQS is limited in the search results, important insights can be drawn from related archaeal enzymes:

• Metal ion preference: M. maripaludis enzymes often show a strong preference for Co^2+ over other divalent metals. For instance, M. maripaludis mmp-RNase Z exhibits 2,990-fold enhanced activity with Co^2+ compared to other metals. This suggests DHQS from the same organism may also have evolved a strong Co^2+ preference.

• Comparative metal preferences:

• NAD+ binding: As DHQS utilizes NAD+ as a redox cofactor for the oxidation step of its complex reaction mechanism, the enzyme likely possesses a conserved NAD+ binding domain. The binding affinity and specificity for NAD+ versus NADP+ would be important characteristics to determine.

• Synergistic effects: The interaction between metal binding and NAD+ binding may involve conformational changes that optimize the active site geometry. Research with other DHQS enzymes suggests these factors work cooperatively rather than independently.

• Experimental determination: To characterize these effects, activity assays should be performed with varying concentrations of different metal ions (Co^2+, Mg^2+, Mn^2+, Zn^2+) and NAD+, both independently and in combination.

What is the impact of temperature and pH on DHQS activity and stability?

• Temperature effects:

  • Activity optimum: Likely around 35-40°C, corresponding to the organism's growth temperature

  • Thermal stability: DHQS from M. maripaludis would be expected to show moderate thermostability, less than hyperthermophilic archaeal enzymes but potentially greater than bacterial homologs

  • Comparative assay temperatures: While M. maripaludis enzyme assays are typically performed at 37°C , assays for DHQS from the hyperthermophilic archaeon Pyrococcus were conducted at 60°C , reflecting their different thermal optima

• pH effects:

  • The standard assay buffer for DHQS activity uses potassium-PIPES buffer at pH 7.0 , suggesting an optimal pH in the neutral range

  • Complete pH profiles should be determined using appropriate buffer systems (MES, PIPES, HEPES, Tris) across pH 5.0-9.0

  • pH stability may differ from pH optimum and should be separately characterized by pre-incubation studies

• Combined effects:

  • Temperature and pH optima often show interdependence

  • At elevated temperatures, the pKa values of ionizable groups can shift, potentially altering pH optima

  • Experimental design should include multifactorial analysis of temperature and pH effects

• Stabilizing factors:

  • Addition of glycerol (5-20%)

  • Presence of cofactors (NAD+)

  • Optimal metal ion concentration (likely Co^2+)

  • Reducing agents to prevent oxidative damage

How does DHQS function within the broader context of aromatic amino acid metabolism in M. maripaludis?

M. maripaludis possesses a fascinating metabolic network for aromatic amino acid biosynthesis that includes multiple pathways with distinct evolutionary origins:

• Integration with canonical shikimate pathway: DHQS (MMP0006) catalyzes an early step in the classical pathway leading to chorismate, the precursor for all three aromatic amino acids. Genetic evidence demonstrates this pathway's essentiality when alternative sources are unavailable, as deletion of DHQ dehydratase results in complete aromatic amino acid auxotrophy .

• Cross-talk with alternative pathways: M. maripaludis can utilize exogenous aryl acids via indolepyruvate oxidoreductase (IOR) to synthesize aromatic amino acids, bypassing the need for de novo synthesis . The organism can satisfy its aromatic amino acid requirements through phenylacetate, indoleacetate, and p-hydroxyphenylacetate when the shikimate pathway is disrupted .

• Novel DKFP pathway: Recent research has identified a unique 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for aromatic amino acid and p-aminobenzoic acid (PABA) biosynthesis that operates independently of chorismate . Notably, PABA was shown to be derived from an early intermediate in this pathway rather than from chorismate as in other organisms .

• Regulatory mechanisms: DKFP pathway activity is reduced when cells are grown with aryl acids , indicating sophisticated regulatory cross-talk between these metabolic routes. This suggests that M. maripaludis has evolved mechanisms to detect substrate availability and adjust its metabolic fluxes accordingly.

• Evolutionary implications: The presence of multiple routes for aromatic amino acid biosynthesis suggests interesting evolutionary dynamics and potential metabolic advantages in fluctuating environments.

How can structural analysis enhance our understanding of archaeal DHQS function?

Structural characterization of M. maripaludis DHQS would provide valuable insights into its unique features and catalytic mechanism:

• Comparative structural analysis: While specific structural information about MMP0006 is not provided in the search results, comparison with DHQS structures from other organisms could reveal:

  • Archaeal-specific structural adaptations

  • Metal binding site architecture that may explain the preference for Co^2+ observed in other M. maripaludis enzymes

  • NAD+ binding domain organization

  • Substrate recognition features

• Catalytic mechanism insights: DHQS catalyzes a complex multistep reaction involving:

  • Alcohol oxidation

  • β-elimination of phosphate

  • Carbonyl reduction

  • Intramolecular aldol condensation

  Structural studies with substrate analogs or reaction intermediates could elucidate how a single active site coordinates these diverse chemical transformations.

• Quaternary structure determination: DHQS enzymes from different organisms exhibit various quaternary structures (monomers, dimers, tetramers). The oligomeric state of M. maripaludis DHQS would have implications for:

  • Enzyme stability

  • Potential allosteric regulation

  • Evolution and adaptation to the archaeal cellular environment

• Structure-guided engineering: Detailed structural information would facilitate:

  • Rational design of more stable variants

  • Engineering enzymes with altered substrate specificity

  • Development of specific inhibitors for antimicrobial applications

• Recommended structural biology approaches:

  • X-ray crystallography of the enzyme with bound cofactors, substrates, or substrate analogs

  • Cryo-electron microscopy for quaternary structure analysis

  • Hydrogen-deuterium exchange mass spectrometry to probe dynamics

  • Molecular dynamics simulations to understand conformational changes during catalysis

What transcriptomic and proteomic approaches can reveal regulation of DHQS expression?

Comprehensive omics approaches provide powerful tools for understanding DHQS regulation in the context of M. maripaludis metabolism:

• Transcriptomic analysis: RNA sequencing can reveal expression patterns of MMP0006 under different growth conditions:

  • Various carbon sources

  • Presence/absence of aromatic amino acids or aryl acids

  • Different growth phases

  • Stress conditions

  The search results describe transcriptomic methods including RNA isolation, quality assessment using spectrophotometry, and sequencing using the Solexa Genome Analyzer . Gene expression can be calculated using the transcripts per million (TPM) method, and comparative analysis can identify differentially expressed genes .

• Ribosomal and translational regulation: Studies with recombinant protein-expressing E. coli revealed upregulation of ribosome-related genes (including rplO, rpsK, rplV, rplP, rpsD, rplR, rpsC, rpsE, and rplB) and aminoacyl-tRNA biosynthesis genes . Similar analyses in M. maripaludis could reveal translational regulation of DHQS.

• Proteomic approaches: Quantitative proteomics can determine:

  • DHQS protein levels under different conditions

  • Post-translational modifications affecting enzyme activity

  • Protein-protein interactions that may regulate DHQS

  • Turnover rates and stability of the enzyme

• Metabolic flux analysis: Isotopic labeling combined with metabolomics can map carbon flow through:

  • The canonical shikimate pathway involving DHQS

  • The alternative DKFP pathway

  • The aryl acid incorporation pathway

  This approach could reveal how M. maripaludis regulates flux through competing pathways. Metabolic labeling with [U-^(13)C]-acetate has been successfully applied to track carbon flow through the DKFP pathway .

• Systems biology integration: Combining transcriptomic, proteomic, and metabolomic data would provide a comprehensive view of how DHQS functions within the broader metabolic network of M. maripaludis and how its expression is coordinated with other enzymes in aromatic amino acid biosynthesis.

What are the potential biotechnological applications of recombinant M. maripaludis DHQS?

Recombinant M. maripaludis DHQS offers several promising biotechnological applications:

• Biocatalysis and green chemistry:

  • DHQS catalyzes a complex multistep reaction with stereospecificity that would be challenging for traditional chemical synthesis

  • Engineered variants could potentially accept non-natural substrates to produce novel compounds

  • Integration into enzymatic cascades for the production of high-value aromatics and pharmaceuticals

• Metabolic engineering applications:

  • Implementation of the archaeal shikimate pathway in heterologous hosts for enhanced aromatic compound production

  • Engineering microorganisms with M. maripaludis DHQS for improved thermostability or metal tolerance

  • Development of synthetic biology platforms incorporating archaeal metabolic modules

• Structural biology and drug discovery:

  • DHQS is absent in mammals but essential in many microorganisms, making it a potential antimicrobial target

  • Structural information from archaeal DHQS could guide the design of selective inhibitors

  • Comparative analysis with bacterial DHQS could reveal unique features for targeting specific pathogens

• Protein engineering opportunities:

  • Directed evolution for enhanced thermostability, solvent tolerance, or catalytic efficiency

  • Creation of fusion proteins combining DHQS with other shikimate pathway enzymes

  • Development of biosensors based on DHQS activity for metabolic engineering applications

• Educational applications:

  • DHQS offers an excellent model system for teaching concepts in enzymology, metabolism, and protein structure-function relationships

  • The complex reaction mechanism provides opportunities to explore fundamental principles in biocatalysis