Recombinant Pseudomonas putida Probable malate:quinone oxidoreductase 3 (mqo3), partial

What is the biochemical function of malate:quinone oxidoreductase 3 in P. putida metabolism?

Malate:quinone oxidoreductase 3 (mqo3) in P. putida catalyzes the oxidation of malate to oxaloacetate coupled with the reduction of quinone to quinol. Unlike conventional NAD-dependent malate dehydrogenases, mqo3 directly feeds electrons into the respiratory chain, contributing to P. putida's versatile metabolism. This enzyme plays a critical role in the tricarboxylic acid (TCA) cycle and directly contributes to cellular bioenergetics .

The reaction can be represented as:
L-malate + quinone → oxaloacetate + quinol

This reaction is particularly important in P. putida due to its versatile metabolism with diverse intrinsic enzymatic capacities, making it an excellent host for heterologous expression of various biosynthetic pathways .

How does mqo3 compare structurally with MQO enzymes from other organisms?

While the specific structure of P. putida mqo3 has not been fully characterized, comparative analysis with other MQO enzymes provides valuable insights. MQO enzymes typically contain:

• A FAD-binding domain with a Rossmann fold motif

• A substrate-binding pocket containing conserved catalytic residues

• A quinone-binding site

Based on characterized MQOs from other organisms such as P. falciparum, the active site likely includes:

• A histidine residue (similar to PfMQO-H123) that functions as a proton/hydride mediator during catalysis

• Tyrosine and additional histidine residues (similar to PfMQO-Y330 and H343) that facilitate correct substrate binding

• An arginine residue (similar to PfMQO-R446) that likely interacts with the C4-carboxyl group of malate

The catalytic mechanism likely involves proton/hydride transfer mediated by a histidine residue in the shallow area near the tunnel entrance of the enzyme .

What expression systems work best for recombinant P. putida mqo3 production?

For optimal recombinant expression of mqo3 in P. putida, several expression systems have proven effective:

• XylS/Pm expression system:

  • Inducible by 3-methylbenzoate (3-mBz)

  • Provides tight inducer-dependent regulation

  • Offers high expression levels

  • Compatible with plasmids pS438·MKc and MKs1

• RhaRS/PrhaBAD expression system:

  • Inducible by rhamnose

  • Features tight regulation with minimal basal expression

  • Suitable for proteins that may be toxic when overexpressed

  • Compatible with plasmids pS4318·MKc and MKs1

Both systems are particularly effective due to their reportedly tight inducer-dependent regulation and high levels of gene expression. For heterologous expression in E. coli or other hosts, codon optimization may be necessary to account for P. putida's high GC content .

What purification protocol yields active recombinant mqo3?

Based on successful purification of MQO proteins from other organisms, the following protocol is recommended for P. putida mqo3:

• Cell lysis:

  • Mechanical disruption (French press or sonication) in buffer containing 50 mM MOPS (pH 7.0), 10% glycerol, and protease inhibitors

  • Addition of detergent (1% Triton X-100 or n-dodecyl β-D-maltoside) to solubilize membrane-associated protein

• Purification steps:

  • Affinity chromatography using Ni-NTA for His-tagged protein

  • Ion exchange chromatography (Resource Q) to remove impurities

  • Size exclusion chromatography for final polishing

• Stabilization considerations:

  • Maintain FAD cofactor association by supplementing all buffers with 10-50 μM FAD

  • Include 10% glycerol to enhance protein stability

  • Maintain a pH of 7.0-7.5 throughout purification

For optimal activity, the purified enzyme should be assessed immediately using a spectrophotometric activity assay measuring the reduction of quinones .

What are the optimal conditions for measuring mqo3 activity?

For reliable measurement of P. putida mqo3 activity, the following conditions have proven effective based on studies of MQO enzymes:

Spectrophotometric Assay Conditions:

• Buffer: 50 mM MOPS, pH 7.0 (optimal pH for MQO activity)

• Temperature: 37°C (physiological temperature)

• Electron acceptors: Ubiquinones of varying side chain length (UQ0, UQ1, UQ2, dUQ)

• Substrate: L-malate (typically 0.5-50 mM range for kinetic studies)

• Additional components: 1 mM KCN (to inhibit downstream electron transport)

• Measurement: Decrease in absorbance at 278 nm (ε = 12,000 M⁻¹ cm⁻¹) corresponding to quinone reduction

Alternatively, a coupled assay system using dichlorophenolindophenol (DCIP) can be employed:

• 120 μM DCIP in assay buffer

• 2 μM antimycin A (to inhibit complex III)

• 60 μM decylubiquinone

• 10 mM L-malate

• Monitoring DCIP reduction at 600 nm (ε = 21,000 M⁻¹ cm⁻¹)

What kinetic parameters characterize mqo3 catalytic activity?

Based on studies of MQO enzymes from other organisms, P. putida mqo3 likely exhibits the following kinetic parameters:

Table 1: Expected Kinetic Parameters for P. putida mqo3

These parameters suggest:

• Moderate affinity for malate (Km likely between CjMQO and PfMQO values)

• Higher affinity for quinones with longer side chains (until solubility becomes limiting)

• Substrate inhibition at higher concentrations of UQ2 and dUQ (>10 μM)

The enzymatic reaction likely follows a ping-pong mechanism similar to other characterized MQO enzymes, with reduction of the FAD cofactor by malate followed by reoxidation by the quinone substrate .

How can computational approaches assist in mqo3 structure prediction?

Computational approaches provide valuable insights into mqo3 structure in the absence of crystallographic data:

• Homology modeling:

  • Using solved structures of homologous MQO enzymes as templates

  • Tools like SWISS-MODEL or Phyre2 can generate initial models

  • Refinement using molecular dynamics simulations

• AI-based structure prediction:

  • AlphaFold has been successfully used for predicting MQO structures

  • The accuracy of computational predictions can be validated using protein footprinting techniques like acetylation with acetic anhydride

• Active site identification:

  • Combining computational prediction with site-directed mutagenesis

  • Conservation analysis across MQO family members identifies potentially crucial residues

  • Molecular docking of substrates (malate) and inhibitors (ferulenol, embelin) to predict binding sites

A comprehensive approach combining these computational methods with experimental validation through site-directed mutagenesis provides the most reliable structural insights into mqo3.

What mutagenesis approaches are most effective for studying mqo3 function?

Based on studies of homologous MQO enzymes, the following mutagenesis approaches are recommended:

• Alanine scanning of conserved residues:

  • Target histidine residues potentially involved in proton/hydride transfer

  • Substitute conserved arginine residues that may interact with malate's carboxyl groups

  • Modify tyrosine residues potentially involved in substrate binding

• Domain swapping:

  • Exchange domains between mqo3 and other MQO family members to identify functional regions

  • Create chimeric proteins to investigate substrate specificity determinants

• Site-directed mutagenesis targeting specific functions:

  • Modify residues in the putative FAD-binding site to assess cofactor interaction

  • Alter membrane-association domains to study localization effects

  • Introduce mutations that may enhance inhibitor sensitivity (e.g., similar to mutations that enhance sensitivity to ferulenol)

These approaches should be coupled with detailed kinetic analysis and, when possible, structural studies to correlate mutations with specific functional changes.

How can mqo3 be incorporated into metabolic engineering of P. putida?

The integration of mqo3 into metabolic engineering strategies for P. putida offers several advantages:

• TCA cycle optimization:

  • Overexpression of mqo3 can enhance TCA cycle flux

  • This may increase precursor availability for biosynthetic pathways

  • Careful balancing with other TCA enzymes is necessary to avoid metabolic imbalances

• Electron transport chain engineering:

  • Modified mqo3 variants can be used to redirect electron flow

  • This approach may enhance energetic efficiency for specific bioprocesses

  • Coupling with ubiquinone biosynthesis engineering for optimized redox balance

• Adaptive laboratory evolution (ALE) targeting:

  • Using automated DIY frameworks for ALE experiments with P. putida

  • Specifically targeting mqo3 function during adaptation to new carbon sources

  • Implementation of dual-chamber semi-continuous bioreactors for long-term evolution experiments

• Integration with heterologous pathway expression:

  • Coordinating mqo3 expression with introduced biosynthetic pathways

  • Leveraging P. putida's versatile metabolism and tolerance to xenobiotics

  • Utilizing mqo3 to support high-yield production of valuable natural products

P. putida's natural properties, including remarkable solvent tolerance and versatile metabolism, make it an ideal chassis for metabolic engineering applications involving mqo3 .

What role does mqo3 play in P. putida adaptation to different carbon sources?

Understanding mqo3's role in carbon source adaptation is crucial for metabolic engineering applications:

• Adaptation mechanisms:

  • mqo3 expression levels change in response to carbon source availability

  • The enzyme facilitates metabolic flexibility by maintaining TCA cycle function under varying conditions

  • Genomic changes affecting mqo3 regulation have been observed during adaptation to new carbon sources

• Experimental approaches to study adaptation:

  • Automated frameworks for adaptive laboratory evolution

  • Long-term cultivation experiments (e.g., 42-day protocols of iterative regrowth)

  • Genomic analysis of evolved populations to identify mutations affecting mqo3 function

• Carbon source-specific considerations:

  • mqo3 activity becomes particularly important during growth on C2 and C3 compounds

  • The enzyme helps balance redox state during metabolism of aromatic compounds

  • Different isozymes may be preferentially expressed depending on carbon source availability

How do known MQO inhibitors interact with mqo3 and what is their specificity?

Understanding inhibitor interactions provides insights into mqo3 function and potential applications:

• Known MQO inhibitors:

  • Ferulenol: A submicromolar inhibitor of mitochondrial MQO that also inhibits bacterial MQOs

  • Embelin: A nanomolar inhibitor showing mixed-type inhibition versus malate

  • Both inhibitors demonstrate noncompetitive inhibition versus quinone

• Inhibition mechanisms:

  • Mixed-type inhibition versus malate suggests the existence of a third binding site to accommodate these inhibitors

  • This trait appears conserved between mitochondrial and bacterial MQOs

  • Inhibitor binding likely induces conformational changes affecting substrate binding

• Specificity considerations:

  • MQO is not conserved in mammals, making it an attractive drug target

  • Species-specific differences in inhibitor sensitivity can be exploited

  • Single substitution mutations near the catalytic site can enhance sensitivity to inhibitors like ferulenol

Table 2: Inhibition Characteristics of Key MQO Inhibitors

These inhibitor studies provide valuable insights into mqo3 catalytic mechanism and potential applications in antimicrobial development .

How can mqo3 be leveraged as a potential antibiotic target?

The potential of mqo3 as an antibiotic target stems from several factors:

• Essentiality for bacterial survival:

  • MQO enzymes have been shown to be essential for the survival of several bacteria

  • Inhibition of MQO activity can inhibit in vitro growth of pathogenic bacteria like C. jejuni

• Target validation approaches:

  • Genetic knockout studies to confirm essentiality

  • Growth inhibition correlation with enzyme inhibition

  • Demonstration of target engagement in cellular contexts

• Drug discovery strategies:

  • Structure-based design leveraging computational models

  • High-throughput screening against purified recombinant mqo3

  • Fragment-based approaches to identify novel chemical scaffolds

  • Rational modification of known inhibitors like ferulenol and embelin

• Advantages as a drug target:

  • Not conserved in mammals, reducing potential toxicity

  • Involved in central metabolism, making resistance development less likely

  • Conservation across multiple bacterial species, offering broad-spectrum potential

The proven inhibition of bacterial growth by MQO inhibitors supports its potential as an antibiotic target, though further validation in specific pathogens is necessary .

How can adaptive laboratory evolution enhance mqo3 expression and function?

Adaptive laboratory evolution (ALE) offers powerful approaches to enhance both mqo3 expression and function:

• Experimental setup for ALE with P. putida:

  • Dual-chamber semi-continuous log-phase bioreactor design

  • Anti-biofilm layout to manage specific traits during long-term cultivation

  • Automated systems for iterative regrowth protocols (typical duration: 42 days)

• Selection strategies:

  • Gradually increasing selective pressure (e.g., decreasing substrate concentration)

  • Alternating selection conditions to enhance robustness

  • Monitoring mqo3 activity throughout the evolution process

• Genomic analysis of evolved strains:

  • Whole-genome sequencing to identify beneficial mutations

  • Specific attention to mutations affecting mqo3 regulation

  • Identification of RNA polymerase mutations that may influence global gene expression patterns

• Implementation of beneficial mutations:

  • Reconstruction of identified mutations in clean genetic backgrounds

  • Combination of beneficial mutations for synergistic effects

  • Integration of evolved mqo3 variants into production strains

ALE approaches have successfully generated P. putida strains with improved growth on non-native carbon sources, with genomic changes revealing the role of RNA polymerase in controlling physiological conditions - similar approaches can be applied specifically to enhance mqo3 function .

What challenges exist in harmonizing multi-omics data for mqo3 research?

Effective mqo3 research requires integration of data from multiple omics technologies:

• Challenges in multi-omics data integration:

  • Different omics platforms generate diverse data formats

  • Temporal alignment of datasets collected at different metabolic states

  • Integration of membrane protein data (like mqo3) which may be underrepresented in some analyses

  • Connecting transcriptomic changes with metabolic flux alterations

• Harmonization strategies:

  • Standardized protocols across omics platforms

  • Development of consistent data formats and quality control measures

  • Implementation of bioinformatic pipelines specifically designed for integration

  • Application of machine learning approaches for pattern recognition across datasets

• Benefits of harmonized multi-omics for mqo3 research:

  • Comprehensive understanding of mqo3's role in cellular metabolism

  • Identification of regulatory networks controlling mqo3 expression

  • Correlation of mqo3 activity with global metabolic state

  • Enhanced reproducibility of research findings across laboratories

Harmonized multi-omics approaches provide a holistic view of mqo3 function within the complex metabolic network of P. putida, enabling more effective metabolic engineering strategies and fundamental insights into enzyme function .

How can recombinant mqo3 be leveraged for biocatalytic applications?

The unique properties of mqo3 offer several opportunities for biocatalytic applications:

• Potential biocatalytic reactions:

  • Selective oxidation of α-hydroxy acids to α-keto acids

  • Regeneration of quinone cofactors for other enzymatic processes

  • Integration into multi-enzyme cascade reactions

• Advantages of mqo3 as a biocatalyst:

  • Cofactor independence (unlike NAD-dependent dehydrogenases)

  • Ability to directly couple with electron transport systems

  • Stability in the presence of organic solvents (leveraging P. putida's solvent tolerance)

  • Potential for operation in two-phase systems

• Engineering strategies for enhanced biocatalysis:

  • Directed evolution for altered substrate specificity

  • Protein engineering to enhance stability in reaction conditions

  • Immobilization techniques for increased reusability

  • Integration with other enzymes for cascade reactions

• Potential applications:

  • Fine chemical synthesis

  • Pharmaceutical intermediate production

  • Bioremediation of specific pollutants

  • Biosensing applications for malate detection

Leveraging P. putida's inherent advantages, including its versatile metabolism and exceptional tolerance to xenobiotics, makes mqo3-based biocatalysis particularly promising for reactions involving challenging substrates or conditions .

How can P. putida mqo3 contribute to sustainable bioproduction processes?

The integration of mqo3 into sustainable bioproduction processes offers several advantages:

• Role in bioconversion processes:

  • Contribution to efficient TCA cycle operation during bioconversion

  • Support for high-yield production of natural products

  • Enhancement of carbon efficiency through improved metabolic flux

• Integration with renewable feedstock utilization:

  • Supporting metabolism of diverse carbon sources derived from biomass

  • Enhancing TCA cycle function during mixed substrate utilization

  • Maintaining redox balance during challenging feedstock metabolism

• Contribution to P. putida's advantages as a production host:

  • Support for P. putida's versatile intrinsic metabolism

  • Contribution to the "clean" metabolic background that simplifies detection of heterologously synthesized metabolites

  • Enhancement of P. putida's tolerance to xenobiotics and solvents

• Specific application examples:

  • Recombinant rhamnolipid production (replacing pathogenic P. aeruginosa)

  • Terpenoid biosynthesis leveraging TCA cycle intermediates

  • Polyketide and non-ribosomal peptide production

  • Other amino acid-derived compounds

P. putida's emergence as a microbial laboratory workhorse, combined with specific advantages for natural product biosynthesis, positions mqo3-enhanced strains as valuable platforms for sustainable bioproduction .