Recombinant Comamonas testosteroni Quinoline 2-oxidoreductase beta chain

What is the structural composition of Quinoline 2-oxidoreductase from Comamonas testosteroni?

Quinoline 2-oxidoreductase from Comamonas testosteroni is a complex enzyme with a native molecular mass of approximately 360 kDa. The enzyme consists of three non-identical subunits with molecular masses of 87 kDa, 32 kDa, and 22 kDa, occurring in a ratio of 1.16:1:0.83 . The 32 kDa subunit corresponds to the beta chain of the enzyme. This heterotrimeric structure is characteristic of molybdoenzymes in the molybdo-iron/sulfur flavoprotein family. The enzyme contains FAD, molybdenum, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8 . Molybdopterin cytosine dinucleotide serves as the organic part of the pterin molybdenum cofactor, which is crucial for the enzyme's catalytic function .

What is the primary reaction catalyzed by Quinoline 2-oxidoreductase?

Quinoline 2-oxidoreductase catalyzes the hydroxylation of quinoline to 2-oxo-1,2-dihydroquinoline. Additionally, it catalyzes the hydroxylation of 3-methylquinoline to 3-methyl-2-oxo-1,2-dihydroquinoline . This reaction represents the first step in the bacterial degradation pathway of quinoline and its derivatives. The enzyme specifically adds a hydroxyl group at the 2-position of the quinoline ring, which is subsequently followed by ring cleavage reactions in the catabolic pathway. The second step in this pathway involves 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase, which performs dioxygenation at the benzene ring .

How conserved is the Quinoline 2-oxidoreductase beta chain across different bacterial species?

The beta-subunits of Quinoline 2-oxidoreductases show remarkable conservation across different bacterial species, particularly at their N-terminal regions. Specifically, high sequence similarity has been observed between the beta-subunits from Comamonas testosteroni 63, Pseudomonas putida 86, Rhodococcus spec. B1, and the related enzyme quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec . This conservation suggests the beta chain plays a crucial functional role that has been maintained through evolution. The conservation is particularly notable in regions likely involved in cofactor binding and electron transfer functions, highlighting the importance of these domains for enzymatic activity.

What metabolic role does Quinoline 2-oxidoreductase play in Comamonas testosteroni?

Quinoline 2-oxidoreductase participates in distinctive catabolic pathways that enable Comamonas testosteroni to grow on various aromatic compounds as the sole source of carbon and energy . Specifically, it catalyzes the initial step in quinoline degradation, converting quinoline to 2-oxo-1,2-dihydroquinoline. This hydroxylation reaction is crucial for ring-opening in subsequent metabolic steps. Comamonas species, including C. testosteroni, are known for their ability to catabolize a wide range of organic and inorganic substrates, making them ecologically important in aquatic and soil environments, including wastewater systems . Their metabolic versatility contributes to the natural biodegradation of aromatic pollutants in these ecosystems.

What is known about electron transfer in Quinoline 2-oxidoreductase?

The electron transfer system in Quinoline 2-oxidoreductase involves multiple redox centers distributed across the enzyme complex. In related quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni, the shortest distance between pyrroloquinoline quinone and heme c is 12.9 Å, which represents one of the longest physiological edge-to-edge distances yet determined between two redox centers . A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers and appears essential for electron transfer . Additionally, a water channel delineates a possible pathway for proton transfer from the active site to the solvent . In Quinoline 2-oxidoreductase, electron transfer likely involves the molybdenum center, iron-sulfur clusters, and FAD cofactors working in concert to catalyze substrate hydroxylation.

What expression systems are most effective for producing recombinant Quinoline 2-oxidoreductase beta chain?

Expression of recombinant Quinoline 2-oxidoreductase beta chain presents several challenges due to its cofactor requirements and involvement in a multi-subunit complex. Based on studies with similar molybdoenzymes, bacterial expression systems, particularly E. coli strains like BL21(DE3) or Rosetta(DE3), have shown the most promise when combined with specialized vectors.

For optimal expression, researchers should consider the following methodology:

• Vector selection: pET vectors with T7 promoter systems offer strong, inducible expression

• Host strain optimization: E. coli strains with enhanced capacity for disulfide bond formation (e.g., Origami) may improve proper folding

• Growth conditions: Post-induction growth at lower temperatures (16-20°C) often improves solubility

• Media supplementation: Addition of iron salts and sodium molybdate can enhance cofactor incorporation

• Co-expression strategies: Consider co-expressing with chaperones or partner subunits to improve folding

A systematic expression optimization table might include:

What analytical techniques are most effective for characterizing the interaction between the beta chain and other subunits?

Understanding subunit interactions is crucial for elucidating the assembly and function of the complete Quinoline 2-oxidoreductase complex. Several complementary techniques can provide valuable insights:

Biophysical approaches:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine precise molecular weights and stoichiometries of complexes

• Isothermal titration calorimetry (ITC) provides thermodynamic parameters (ΔH, ΔS, Kd) of subunit binding

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) measures binding kinetics (kon, koff)

• Analytical ultracentrifugation (AUC) characterizes complex formation under native conditions

Structural approaches:

Functional approaches:

• Activity assays comparing individual subunits versus reconstituted complexes

• Mutagenesis of putative interface residues followed by binding and activity analysis

• Co-purification experiments to assess complex stability under various conditions

The combination of these methods can generate a comprehensive model of how the beta chain interacts with other subunits and contributes to the enzyme's quaternary structure and function.

How does the beta chain contribute to cofactor binding and electron transfer?

The beta chain of Quinoline 2-oxidoreductase likely plays a crucial role in coordinating iron-sulfur clusters that facilitate electron transfer between redox centers. Based on studies of related enzymes, the following methodological approaches can help elucidate these functions:

Spectroscopic analysis:

• Electron paramagnetic resonance (EPR) spectroscopy to characterize iron-sulfur cluster types and redox properties

• Magnetic circular dichroism (MCD) to probe the electronic structure of metal centers

• Resonance Raman spectroscopy to examine iron-sulfur cluster coordination environments

• UV-visible spectroscopy to monitor changes in cofactor oxidation states during catalysis

Site-directed mutagenesis:

• Mutation of conserved cysteine residues that likely coordinate iron-sulfur clusters

• Analysis of resulting changes in spectroscopic properties, cofactor content, and catalytic activity

• Identification of residues involved in electron transfer pathways through systematic mutagenesis

Redox potentiometry:

• Determination of reduction potentials of individual redox centers

• Construction of redox potential maps to understand electron flow directionality

• Comparison of wild-type and mutant proteins to assess the impact of specific residues

Understanding the beta chain's role in electron transfer is essential, as the efficiency of this process directly affects the enzyme's catalytic rate and substrate conversion in hydroxylation reactions.

What are the optimal conditions for purifying recombinant Quinoline 2-oxidoreductase beta chain while maintaining activity?

Purification of recombinant Quinoline 2-oxidoreductase beta chain requires careful attention to maintaining protein stability and cofactor integrity. A comprehensive purification strategy should include:

Buffer optimization:

• pH range: 7.0-8.0 (typical optimum for molybdoenzymes)

• Buffer components: 50 mM phosphate or Tris with 150-300 mM NaCl

• Reducing agents: 2-5 mM DTT or 5-10 mM β-mercaptoethanol to maintain sulfhydryl groups

• Protease inhibitors: PMSF, leupeptin, pepstatin A, or commercial cocktails

Chromatographic strategy:

• Affinity chromatography (if tagged): IMAC for His-tagged protein with gradient elution

• Ion exchange chromatography: Typically anion exchange at pH 7.5-8.0

• Size exclusion chromatography: Final polishing step and assessment of oligomeric state

Activity preservation:

• Inclusion of stabilizing agents: 10-20% glycerol, 0.1% Triton X-100

• Addition of cofactor precursors: sodium molybdate (1-10 μM), ferrous ammonium sulfate (10-50 μM)

• Low temperature operations (4°C where possible)

• Avoid freeze-thaw cycles; store at -80°C with cryoprotectants

A typical purification table might show:

How can enzyme kinetics distinguish the mechanistic contributions of the beta chain in the catalytic process?

Kinetic analysis can provide valuable insights into the specific contributions of the beta chain to Quinoline 2-oxidoreductase catalysis. A comprehensive kinetic investigation should include:

Steady-state kinetics:

• Determination of kinetic parameters (Km, kcat, kcat/Km) for the full complex versus complexes with modified beta chains

• Analysis of pH and temperature dependence to identify ionizable groups and activation parameters

• Substrate specificity studies using various quinoline derivatives to map active site constraints

Pre-steady-state kinetics:

• Stopped-flow spectroscopy to resolve individual steps in the catalytic cycle

• Rapid-quench techniques to identify reaction intermediates

• Burst kinetics analysis to identify rate-limiting steps

Electron transfer kinetics:

• Use of artificial electron acceptors with different redox potentials

• Measurement of electron transfer rates using spectroscopic techniques

• Comparison of wild-type beta chain with mutated versions lacking specific redox centers

Inhibition studies:

• Competitive inhibitors to probe active site accessibility

• Metal chelators to assess the role of different metal centers

• Specific inhibitors of electron transfer to isolate this contribution

What structural techniques provide the most valuable insights into the beta chain's three-dimensional architecture?

Understanding the three-dimensional structure of the Quinoline 2-oxidoreductase beta chain is crucial for elucidating its function. Several complementary structural biology techniques provide valuable insights:

X-ray crystallography:

• Provides atomic-level resolution (potentially 1.5-2.5 Å) of protein structure

• Challenges include obtaining diffraction-quality crystals of the beta chain

• Methodological approach: Screening multiple crystallization conditions with purified protein; considering surface entropy reduction or crystallization chaperones

Cryo-electron microscopy (cryo-EM):

• Particularly valuable for visualizing the beta chain in the context of the complete enzyme complex

• Can achieve 2-4 Å resolution for well-behaved samples

• Methodological approach: Vitrification of purified enzyme followed by imaging on a high-end electron microscope with direct electron detector

Small-angle X-ray scattering (SAXS):

• Provides low-resolution (10-20 Å) envelope of protein in solution

• Useful for studying conformational changes under different conditions

• Methodological approach: Collecting scattering data on monodisperse protein samples at multiple concentrations

Nuclear magnetic resonance (NMR) spectroscopy:

• Provides information on protein dynamics and residue-level interactions

• Limited by molecular weight (most applicable to domains of the beta chain)

• Methodological approach: Expression of isotopically labeled protein (15N, 13C) followed by multidimensional NMR experiments

The integration of multiple structural techniques provides a more complete picture than any single method alone. For example, crystallography might reveal precise atomic positions, while SAXS and cryo-EM could show how the beta chain is positioned within the larger complex.

How can isotope labeling and spectroscopy enhance our understanding of the reaction mechanism?

Isotope labeling combined with advanced spectroscopic techniques provides powerful tools for elucidating the catalytic mechanism of Quinoline 2-oxidoreductase. Key methodological approaches include:

Oxygen isotope studies:

• Use of 18O2 to track oxygen incorporation into the hydroxylated product

• LC-MS/MS analysis to determine the precise location of labeled oxygen

• Mechanistic implication: Distinguishes between oxygen derived from water versus molecular oxygen

Hydrogen isotope effects:

• Synthesis of deuterated quinoline substrates (particularly at the C2 position)

• Measurement of primary kinetic isotope effects (KIEs) on kcat and kcat/Km

• Mechanistic implication: Large KIEs (>2) would suggest C-H bond breaking in the rate-limiting step

13C and 15N labeling:

• Site-specific incorporation of 13C or 15N into quinoline substrate

• NMR spectroscopy to monitor chemical shift changes during reaction

• Mechanistic implication: Provides information about electronic changes at specific positions

EPR spectroscopy with freeze-quench:

• Rapid freezing of reaction mixtures at various time points

• EPR analysis to detect paramagnetic intermediates (particularly Mo(V) species)

• Mechanistic implication: Characterizes electron distribution during catalysis

A typical experimental design might include:

What computational approaches can predict substrate binding and catalytic mechanisms?

Computational methods offer valuable insights into aspects of enzyme function that are difficult to study experimentally. For Quinoline 2-oxidoreductase beta chain, several computational approaches are particularly relevant:

Homology modeling and molecular docking:

• Construction of a three-dimensional model based on related enzymes with known structures

• Docking of quinoline and derivatives to predict binding modes

• Analysis of protein-substrate interactions to identify key binding residues

• Methodological approach: Use software like MODELLER for homology modeling and AutoDock Vina for substrate docking

Molecular dynamics simulations:

• All-atom simulations to explore protein dynamics and substrate binding

• Identification of water channels and proton transfer pathways

• Calculation of binding free energies using enhanced sampling techniques

• Methodological approach: Perform 100+ ns simulations using AMBER, GROMACS, or NAMD with appropriate force fields

Quantum mechanical calculations:

• QM or QM/MM methods to model the reaction mechanism

• Calculation of transition state energies and structures

• Investigation of electron transfer pathways between redox centers

• Methodological approach: DFT calculations using software like Gaussian or ORCA, focusing on active site and cofactors

Network analysis:

• Identification of residue interaction networks and allosteric pathways

• Prediction of mutations that might enhance activity or stability

• Methodological approach: Use tools like Protein Structure Network analysis or Dynamical Network Analysis

These computational approaches complement experimental studies by providing atomic-level insights into mechanisms and dynamics that may not be directly observable in the laboratory.

How can the quinoline 2-oxidoreductase system be applied in bioremediation of environmental contaminants?

The metabolic versatility of Comamonas testosteroni, particularly its quinoline degradation pathway, makes it a promising candidate for bioremediation applications. Research methodologies to explore this potential include:

Biodegradation studies:

• Assessment of degradation rates for various N-heterocyclic pollutants

• Identification of metabolic bottlenecks in degradation pathways

• Optimization of conditions for maximal degradation efficiency

• Methodological approach: HPLC or LC-MS monitoring of substrate depletion and metabolite formation

Genetic engineering strategies:

• Overexpression of quinoline 2-oxidoreductase to enhance degradation capacity

• Pathway engineering to expand substrate range

• Development of biosensor strains for pollutant detection

• Methodological approach: Construct expression vectors with strong, inducible promoters for key enzymes

Immobilization technologies:

• Immobilization of whole cells or purified enzymes on various supports

• Comparison of free versus immobilized systems for stability and reusability

• Development of bioreactor configurations for continuous operation

• Methodological approach: Test different immobilization matrices (alginate, polyacrylamide, etc.) and reactor designs

Field-scale applications:

• Pilot studies in controlled contaminated environments

• Monitoring of degradation efficiency and ecological impacts

• Assessment of long-term stability and activity

• Methodological approach: Establish test plots with varying bacterial loads and treatment conditions