Recombinant Prochlorococcus marinus subsp. pastoris Probable malate:quinone oxidoreductase (mqo)

What is the role of malate:quinone oxidoreductase (MQO) in Prochlorococcus marinus metabolism?

MQO plays dual roles in both the electron transport chain (ETC) and the tricarboxylic acid (TCA) cycle. Specifically, it catalyzes the oxidation of malate to oxaloacetate while reducing the quinone pool in the ETC, contributing significantly to cellular bioenergetics . This enzyme is particularly important for understanding Prochlorococcus metabolism because:

• It connects respiratory electron transport to central carbon metabolism

• It represents an alternative to NAD-dependent malate dehydrogenase found in most organisms

• It may be essential for Prochlorococcus survival, similar to its role in other bacteria

Given that Prochlorococcus has one of the smallest genomes among photosynthetic organisms (1.66-1.75 MB) , the retention of MQO suggests its essential nature in this organism's streamlined metabolism.

How should recombinant Prochlorococcus MQO be expressed and purified?

While the search results don't provide specific protocols for Prochlorococcus MQO, we can adapt methods used for related bacterial MQOs:

Expression system optimization:

• Use E. coli NiCo21(DE3) as the expression host, which yields His-tagged recombinant proteins with fewer contaminants after affinity purification

• Optimize IPTG concentration to approximately 10 μM for induction, as this concentration resulted in highest specific activity for related bacterial MQOs

• Consider lower temperature expression (18°C) for extended periods (60+ hours), which has been successful for other Prochlorococcus proteins

Purification approach:

• Include OG (octyl glucoside) detergent during purification, as other commonly used detergents like DDM, Triton X-100, and CHAPS resulted in complete loss of enzymatic activity in related bacterial MQOs

• Check FAD content post-purification, as a FAD:protein ratio near 1.0 correlates with highest enzyme activity

• Verify protein integrity through SDS-PAGE and activity assays

The purification should aim for high specific activity, which for related bacterial MQOs has reached 125 ± 5.4 μmol/min/mg .

How can the activity of recombinant Prochlorococcus MQO be measured in vitro?

Standard assay conditions for measuring MQO activity include:

Reaction mixture components:

• Ubiquinone (UQ) or other appropriate quinone electron acceptors

• 1% (v/v) ethanol

• 50 mM MOPS buffer at pH 7.0 (optimize for Prochlorococcus MQO)

• 1 mM KCN (to inhibit interference from other respiratory enzymes)

• 0.2 μg/mL of purified enzyme

Measurement approach:

• Monitor the reduction of quinones through the decrease in absorbance at 278 nm (extinction coefficient: 15 mM⁻¹ cm⁻¹)

• Determine Michaelis-Menten kinetic parameters by varying substrate concentrations:

  • For quinones: 0.1 to 100 μM (with fixed malate concentration at 10 mM)

  • For malate: 0.5 to 50 mM (with fixed quinone concentration)

Kinetic analysis:

• Calculate kinetic parameters using Michaelis-Menten equation

• Generate Lineweaver-Burk plots to determine reaction mechanism

• For inhibitor studies, use mixed-type inhibition equations with nonlinear regression methods

What cofactors are required for MQO activity in Prochlorococcus marinus?

While specific information for Prochlorococcus MQO is not available in the search results, related bacterial MQOs provide insights:

• FAD is the essential cofactor, with one FAD molecule binding per MQO protein molecule

• The FAD:protein ratio strongly correlates with enzyme activity (optimal ratio between 0.93-1.16)

• The FAD cofactor appears to be tightly but non-covalently bound

• When the FAD:protein ratio decreases (e.g., to 0.7), enzyme activity decreases proportionally

• Importantly, unlike some other flavoenzymes, addition of exogenous FAD does not restore activity once the cofactor is lost, indicating an irreversible process

Researchers should therefore ensure careful handling during purification to maintain FAD binding and maximize enzyme activity.

What is known about substrate specificity of Prochlorococcus MQO?

Based on studies of related bacterial MQOs:

Primary substrates:

• L-malate as electron donor

• Various quinones as electron acceptors (ubiquinone derivatives with different side chain lengths have been tested)

Substrate preferences:

• NAD is not an electron acceptor for MQO, distinguishing it from malate dehydrogenase

• The natural direct acceptor is most likely a quinone specific to the organism's electron transport chain

• For marine cyanobacteria like Prochlorococcus, plastoquinone may be an important electron acceptor in vivo

Experimental approach to determine specificity:
Test various quinones (UQ0, UQ1, UQ2, UQ4, and dUQ) with recombinant enzyme to determine which provides optimal activity and physiological relevance .

How might MQO function differ between Prochlorococcus ecotypes adapted to different ocean depths?

Prochlorococcus consists of multiple ecotypes adapted to different light and oxygen regimes across ocean depths:

High-light adapted ecotypes (e.g., MED4):

• Isolated from surface waters (5m depth) where oxygen levels approach saturation

• May have MQO optimized for higher oxygen environments

• Could show different substrate preferences compared to low-light ecotypes

Low-light adapted ecotypes (e.g., SS120, MIT9313):

• Found at depths of 120m or more where light is limited and oxygen levels are lower

• May show alternative electron transport strategies, potentially involving MQO

• Some Prochlorococcus strains in the North Pacific Ocean are deficient in Cytochrome b6f and may utilize novel electron transport mechanisms

Research methodology to investigate these differences:

• Compare recombinant MQO from different ecotypes biochemically (kinetic parameters, substrate preferences)

• Analyze oxygen dependency of enzyme activity

• Integrate findings with metaproteomic data from natural populations at different ocean depths

Studies have shown that Prochlorococcus doesn't photosynthesize below 200m and may become heterotrophic under low oxygen and light conditions , suggesting MQO might function differently in these environments.

How does the oligomeric state of MQO affect its enzymatic properties?

MQO from different bacterial sources exists in various oligomeric states that affect activity:

Observed oligomeric states in related bacterial MQOs:

• Multiple forms identified through native gel electrophoresis: monomer, dimer, tetramer, and higher-order oligomers

• Activity staining reveals that dimeric forms often show higher activity than tetrameric forms

Hypotheses for oligomer-activity relationship:

• The dimeric form may be more stable with better solvent accessibility to substrate binding sites

• The active site might be located at the dimerization interface

• Tetramerization might hide binding sites at the dimer-dimer interface, reducing activity

Recommended analytical approaches:

• High Resolution Clear Native Electrophoresis (hrCNE)

• Blue Native gel electrophoresis (BN-PAGE) with activity staining

• Size exclusion chromatography combined with multiangle light scattering

• Crosslinking mass spectrometry to identify interaction interfaces

Determining the relationship between oligomeric state and activity would provide fundamental insights into MQO function in Prochlorococcus.

How do inhibitors affect Prochlorococcus MQO activity and what does this reveal about its mechanism?

While specific inhibitor studies of Prochlorococcus MQO are not available in the search results, insights from related bacterial MQOs are valuable:

Known inhibitors of bacterial MQOs:

• Ferulenol: nanomolar inhibitor, mixed-type inhibition versus malate, noncompetitive versus quinone

• Embelin: nanomolar inhibitor with similar inhibition mechanism to ferulenol

Mechanistic insights from inhibitor studies:

• The mixed inhibition pattern suggests the existence of a third binding site distinct from substrate binding sites

• This trait appears to be conserved between mitochondrial and bacterial MQOs

• The inhibitors prevent growth of bacteria in vitro, supporting the essential nature of MQO

Protocol for inhibitor mechanism studies:

• Vary inhibitor concentrations (e.g., ferulenol: 0, 0.01, 0.02, 0.04 μM)

• Measure enzyme activity at different substrate concentrations

• Generate Lineweaver-Burk plots and replot slopes and intercepts as functions of inhibitor concentration

• Confirm mechanism using Dixon and Cornish-Bowden plots

This approach would reveal whether Prochlorococcus MQO shares the same inhibitor binding properties as other bacterial MQOs, providing insights into its evolutionary conservation.

How can Prochlorococcus MQO be integrated into metabolic models to understand its role in carbon flux?

Recent metabolic modeling of Prochlorococcus provides a framework for integrating MQO function:

Current metabolic models:

• iSO595: A genome-scale metabolic model for Prochlorococcus MED4 with 595 genes, 802 metabolites, and 994 reactions

• This model has been used to study carbon storage (glycogen) and exudation under different nutrient conditions

Integration strategies:

• Update reaction stoichiometry for MQO in the model based on biochemical characterization

• Incorporate experimentally determined kinetic parameters

• Use flux balance analysis (FBA) with forced carbon and light influx to recapitulate overflow metabolism

• Apply COMETS (Computation of Microbial Ecosystems in Time and Space) to simulate day-night cycles

Research questions addressable with integration:

• How does MQO activity change during day-night cycles?

• What role does MQO play in carbon storage versus exudation under nutrient limitation?

• How do different quinone pools affect electron distribution in the cell?

The model shows that storage of glycogen or exudation of organic acids are favored when growth is nitrogen limited, while exudation of amino acids becomes more likely when phosphate is limiting . MQO likely plays a key role in these metabolic shifts.

How might MQO interact with the photosynthetic apparatus in Prochlorococcus?

Prochlorococcus has a unique photosynthetic apparatus with several distinctive features:

Unique photosynthetic characteristics:

• Uses divinyl derivatives of chlorophyll a and b (Chl a₂ and b₂)

• Different strains show dramatically different pigment ratios (Chl b₂/Chl a₂)

• Some strains possess a novel type of phycoerythrin

• Photosystem components show adaptations to different light conditions

Potential MQO-photosynthesis interactions:

• MQO may interface with photosystem I through the shared quinone pool

• During day-night transitions, MQO activity might shift to accommodate changing redox states

• In low light conditions, MQO may participate in alternative electron transport pathways

Experimental approach to investigate interactions:

• Compare MQO activity in cells grown under different light regimes

• Analyze temporal patterns of MQO expression relative to photosynthesis genes

• Examine how MQO activity responds to transitions between photosynthesis and glycogen depletion

Research shows that the switch from photosynthesis/glycogen storage to glycogen depletion is associated with a redistribution of fluxes from the Entner-Doudoroff to the Pentose Phosphate pathway . MQO likely plays a key role in these metabolic transitions that connect photosynthesis to central carbon metabolism.

What purification challenges are specific to Prochlorococcus MQO?

Based on experience with related bacterial MQOs, researchers should consider:

Membrane association:

• MQO is typically a peripheral membrane protein that can be released with chelators

• Careful solubilization is critical to maintain activity

Detergent sensitivity:

• OG (octyl glucoside) detergent should be maintained during all purification steps

• Other common detergents (DDM, Triton X-100, CHAPS) may result in complete loss of activity

Cofactor retention:

• Monitor FAD:protein ratio throughout purification

• Loss of FAD correlates with irreversible loss of activity

Oligomeric state considerations:

• Multiple oligomeric states may be present

• Activity may vary between different oligomeric forms

A typical purification yield from related bacterial MQOs is approximately 2 mg of highly active enzyme (125 ± 5.4 μmol/min/mg) from a 3.6 L culture .

How can site-directed mutagenesis be used to probe MQO function in Prochlorococcus?

While genetic tools for direct modification of Prochlorococcus are still limited , recombinant expression allows for mutagenesis studies:

Priority residues for mutagenesis:

• FAD binding residues

• Substrate binding sites (malate and quinone)

• Potential third binding site implicated in inhibitor studies

• Residues at oligomerization interfaces

Experimental approach:

• Generate point mutations in recombinant MQO

• Express and purify mutant proteins

• Analyze changes in:

  • Kinetic parameters (Km, Vmax)

  • Substrate specificity

  • Inhibitor sensitivity

  • Oligomeric state

Interpret findings in context of:

• Sequence conservation across Prochlorococcus ecotypes

• Structural models based on related MQOs

• Metabolic models of Prochlorococcus

These studies could reveal which residues are essential for catalysis versus those that may be involved in adaptation to specific ecological niches.

How can isotopic labeling be used to track MQO activity in metabolic flux studies?

Isotopic labeling provides powerful insights into metabolic fluxes involving MQO:

Experimental design:

• Culture Prochlorococcus with ¹³C-labeled bicarbonate or glucose

• Sample at time points during growth or day-night cycles

• Extract metabolites and analyze by LC-MS/MS or NMR

• Quantify isotopomer distribution in TCA cycle intermediates

Key measurements:

• ¹³C enrichment in malate, oxaloacetate, and other TCA intermediates

• Flux ratios between competing pathways

• Temporal changes in flux distribution

Integration with models:

• Use isotopically non-stationary metabolic flux analysis (INST-MFA)

• Constrain flux balance analysis models with measured fluxes

• Compare experimental data with model predictions

This approach would reveal how carbon flows through MQO under different environmental conditions and how this enzyme contributes to the remarkable metabolic efficiency of Prochlorococcus.

How might climate change affect MQO function in oceanic Prochlorococcus populations?

As ocean conditions change with global warming, Prochlorococcus metabolism may be affected:

Climate-related factors to consider:

• Increasing ocean temperatures

• Changing oxygen concentrations (expanding oxygen minimum zones)

• Altered nutrient availability patterns

• Changes in light penetration due to stratification

Research approaches:

• Compare MQO sequences and expression across Prochlorococcus ecotypes from different oceanic regions

• Determine temperature and oxygen dependence of recombinant MQO activity

• Use metabolic models to predict how changed environmental parameters might affect carbon flux through MQO

• Analyze metaproteomic data from ocean regions experiencing different climate change impacts

Prochlorococcus has been found to play a major role in oceanic carbon fixation, responsible for approximately 50% of marine carbon fixation in some regions . Understanding how MQO function might change under climate scenarios is therefore important for predicting future ocean productivity.

Could comparative studies of MQO across different Prochlorococcus ecotypes reveal evolutionary adaptation mechanisms?

Prochlorococcus has diversified into multiple ecotypes adapted to different ocean depths and light regimes:

Comparative approaches:

• Sequence MQO genes from diverse Prochlorococcus ecotypes

• Express and characterize recombinant MQO from representative strains:

  • High-light adapted (e.g., MED4)

  • Low-light adapted (e.g., SS120, MIT9313)

• Correlate biochemical differences with ecological niches

• Use ancestral sequence reconstruction to trace MQO evolution

Expected insights:

• Identification of adaptive mutations affecting substrate affinity, catalytic efficiency, or regulation

• Understanding of how MQO contributes to the ecological success of different ecotypes

• Potential discovery of novel MQO functions in specific environments

This research would contribute to our understanding of molecular adaptation in minimal genomes and the evolution of metabolic efficiency in the most abundant photosynthetic organism on Earth.

How might the unique metabolism of Prochlorococcus inform synthetic biology applications?

Prochlorococcus has evolved remarkable metabolic efficiency with a minimal genome:

Valuable features for synthetic biology:

• Streamlined metabolism with minimal redundancy

• Efficient photosynthesis adaptable to different light regimes

• Carbon concentration mechanisms

• Dynamic glycogen allocation in response to environmental cues

Potential applications:

• Design of minimal photosynthetic chassis organisms

• Engineering metabolic modules for carbon fixation

• Development of biosensors for marine environments

• Creation of synthetic microbial communities with efficient nutrient cycling

MQO-specific applications:

• Engineering electron transport chains with alternative electron acceptors

• Designing metabolic circuits for dynamic carbon allocation

• Creating biocatalysts for specific redox reactions

The insights gained from studying Prochlorococcus MQO could inform more efficient bioenergy systems and carbon capture technologies.

What is the recommended workflow for studying recombinant Prochlorococcus MQO?

Based on successful approaches with related bacterial MQOs, we recommend:

Step 1: Gene cloning and expression

• Clone the MQO gene from Prochlorococcus with an N-terminal His6-tag

• Transform into E. coli NiCo21(DE3)

• Induce with 10 μM IPTG

• Express at 18°C for extended periods (60+ hours)

Step 2: Purification

• Isolate membrane fraction

• Solubilize with OG detergent

• Purify using affinity chromatography

• Verify purity by SDS-PAGE and determine FAD content

Step 3: Biochemical characterization

• Determine optimal pH and temperature

• Measure kinetic parameters for malate and various quinones

• Analyze reaction mechanism using Lineweaver-Burk plots

• Study inhibition patterns with known MQO inhibitors

Step 4: Structural studies

• Analyze oligomeric state using hrCNE and BN-PAGE

• Perform activity staining to identify active oligomeric forms

• Attempt crystallization for structural determination

Step 5: Integration with metabolic models

• Incorporate biochemical parameters into existing Prochlorococcus models (e.g., iSO595)

• Simulate metabolic fluxes under different environmental conditions

This comprehensive workflow would generate valuable insights into the function of MQO in the context of Prochlorococcus metabolism.

What controls should be included when working with recombinant Prochlorococcus MQO?

To ensure reliable results, include these essential controls:

Expression and purification controls:

• Empty vector control to assess endogenous E. coli MQO activity

• Measurement of specific activity in membrane fractions before purification

• SDS-PAGE analysis to verify protein size and purity

• Determination of FAD:protein ratio

Activity assay controls:

• Reactions without enzyme (spontaneous rate)

• Reactions without substrate

• Inclusion of known MQO inhibitors as positive controls

• Heat-inactivated enzyme

Comparative standards:

• Commercial enzymes with known activity when available

• Well-characterized related enzymes (e.g., C. jejuni MQO)

• Different quinone derivatives to establish substrate preference

Stability controls:

• Monitor activity over time at storage conditions

• Test activity after freeze-thaw cycles

• Assess the effect of different buffer compositions

Proper controls will ensure that observed activities are specifically attributable to the Prochlorococcus MQO and not to contaminants or artifacts.

How should researchers approach studying MQO in the context of the complete Prochlorococcus electron transport chain?

To understand MQO's role in the entire electron transport system:

Experimental approaches:

• Reconstitute partial or complete electron transport chains in liposomes

• Measure electron flow using oxygen electrodes or artificial electron acceptors

• Use specific inhibitors to block different components of the chain

• Employ isotopic labeling to track electron flow through different pathways

Key interactions to investigate:

• Relationship between MQO and photosynthetic electron transport

• Competition with other quinone-reducing enzymes

• Interaction with the cytochrome b6f complex (or its absence in some strains)

• Connection to NDH (NADPH dehydrogenase) and PTOX (plastoquinol terminal oxidase)

Integration with systems biology:

• Correlate enzyme activities with proteomic data from different ocean depths

• Compare findings with metabolic model predictions

• Consider day-night cycling effects on electron transport chain composition and activity

This approach would provide a more comprehensive understanding of how MQO functions within the unique electron transport systems of Prochlorococcus adapted to different ocean environments.