Recombinant Acaryochloris marina NAD (P)H-quinone oxidoreductase subunit L

What is Acaryochloris marina NAD(P)H-quinone oxidoreductase subunit L and what makes it biologically significant?

Acaryochloris marina NAD(P)H-quinone oxidoreductase subunit L (UniProt: B0C6G0) is a protein component of the NAD(P)H dehydrogenase I complex from A. marina, a unique cyanobacterium that primarily uses chlorophyll d for photosynthesis rather than chlorophyll a. This adaptation allows A. marina to efficiently use far-red light for photosynthesis, which represents a specialized niche adaptation . The enzyme catalyzes electron transfer reactions in the photosynthetic electron transport chain, similar to other quinone oxidoreductases that perform two-electron reductions of quinones and various organic compounds . Its significance lies in understanding how photosystems have adapted to utilize lower energy light wavelengths while maintaining efficient photochemical reactions despite the approximately 10% lower photon energy available in the far-red spectrum compared to visible light .

How is the gene encoding this protein organized in the A. marina genome?

The gene encoding NAD(P)H-quinone oxidoreductase subunit L is designated as ndhL with the ordered locus name AM1_1349 in the A. marina genome . The protein is expressed as part of the remarkably large 8.3 million base pair genome of A. marina, which is among the largest bacterial genomes sequenced . This expansive genome includes nine single-copy plasmids that code for over 25% of the putative ORFs, with heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements . The ndhL gene appears to be part of the photosynthetic gene cluster, consistent with its role in electron transport processes essential for photosynthetic function. The expression region spans positions 1-69 of the protein sequence, which may have implications for its membrane orientation and function .

What are the primary structural characteristics of this protein?

The NAD(P)H-quinone oxidoreductase subunit L from A. marina is characterized by the following structural features:

• Amino acid sequence: MVVNLILLLGLLGGYLLVMPAITYFYLQKRWYVASSLERGFMYFLVFFFFPSLLLLSPFLNFRPQPRKI

• Length: 69 amino acids in the mature protein

• Hydrophobic profile: The sequence contains multiple hydrophobic stretches (particularly evident in the LILLGLLGGYLLVM segment), suggesting membrane-associated domains typical of proteins involved in electron transport chains

• The enzyme requires FAD as a cofactor for function, consistent with other quinone oxidoreductases that utilize FAD in a substituted enzyme mechanism

• The protein likely forms part of a larger multi-subunit complex, as suggested by its designation as "subunit L" of the larger NAD(P)H dehydrogenase I (NDH-1) complex

The hydrophobic nature of this protein suggests transmembrane localization, which would be consistent with its role in electron transport processes across photosynthetic membranes.

How does the NAD(P)H-quinone oxidoreductase from A. marina differ functionally from similar enzymes in chlorophyll a-containing cyanobacteria?

The NAD(P)H-quinone oxidoreductase from A. marina operates within a unique photosynthetic system that utilizes chlorophyll d instead of chlorophyll a as the primary photosynthetic pigment. This adaptation represents a significant shift in energy harvesting capabilities, as chlorophyll d absorbs at approximately 697 nm in methanol (700-750 nm in vivo), compared to chlorophyll a, which absorbs at approximately 665.2 nm (670-700 nm in vivo) . This ~30 nm difference corresponds to approximately 80 mV (10%) lower photon energy .

Despite this energy difference, A. marina's photosystems drive similar photochemical reactions to those in chlorophyll a-dependent systems. This adaptation requires potential modifications in the electron transport chain components, including the NAD(P)H-quinone oxidoreductase complex. The functional differences may include:

• Modified redox potentials to accommodate the lower energy input

• Structural adaptations in the quinone binding sites

• Altered interaction with electron donors and acceptors specific to the chlorophyll d-based photosystem

Research indicates that despite the global replacement of major photosynthetic pigments, A. marina has incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments . This suggests highly conserved electron transport mechanisms with subtle but critical modifications in components like the NAD(P)H-quinone oxidoreductase complex to maintain efficient electron flow despite the energetic constraints.

What methodological approaches are recommended for studying the kinetics of recombinant A. marina NAD(P)H-quinone oxidoreductase?

Studying the enzyme kinetics of recombinant A. marina NAD(P)H-quinone oxidoreductase requires specialized approaches that account for its membrane association and involvement in electron transport chains. Recommended methodological approaches include:

• Spectrophotometric assays: Monitor the oxidation of NAD(P)H at 340 nm coupled with the reduction of various quinone substrates. This should be conducted both in the presence and absence of potential inhibitors like dicoumarol (known to inhibit similar enzymes) .

• Oxygen consumption measurements: Using oxygen electrode systems to measure enzyme activity through coupling to oxygen-dependent reactions.

• Artificial electron acceptor studies: Employing compounds like dichlorophenolindophenol (DCPIP) or ferricyanide as artificial electron acceptors to bypass the natural electron transport chain.

• Reconstitution in proteoliposomes: For more physiologically relevant studies, reconstituting the purified enzyme into liposomes to recreate the membrane environment.

• Stopped-flow spectroscopy: To capture rapid electron transfer reactions characteristic of these enzymes.

When analyzing kinetic data, researchers should apply appropriate models that account for the potential cooperativity often observed in quinone oxidoreductases, as there is evidence for negative cooperativity in these enzyme families which may be mediated through alterations in protein mobility .

How might researchers investigate the interaction between the NAD(P)H-quinone oxidoreductase and other components of the photosynthetic electron transport chain in A. marina?

Investigating the interactions between A. marina NAD(P)H-quinone oxidoreductase and other photosynthetic components requires integrative approaches:

• Co-immunoprecipitation studies: Using antibodies against the recombinant NAD(P)H-quinone oxidoreductase subunit L to pull down associated proteins from A. marina thylakoid membranes.

• Blue native PAGE: To isolate intact multiprotein complexes and identify components that associate with the NAD(P)H-quinone oxidoreductase.

• Crosslinking mass spectrometry: To capture transient interactions between the enzyme and other electron transport chain components.

• Förster resonance energy transfer (FRET): To examine proximity and dynamic interactions between fluorescently labeled components.

• Cryo-electron microscopy: To visualize the structural organization of the entire complex within the thylakoid membrane context.

• Computational molecular docking: To predict interaction interfaces between the NAD(P)H-quinone oxidoreductase and other proteins like photosystem I (PSI), which has been structurally characterized in A. marina .

These approaches would help elucidate how this enzyme interfaces with A. marina's unique photosystem I, which contains primarily chlorophyll d (approximately 70 chlorophyll d molecules per complex) and α-carotenes instead of β-carotenes found in typical cyanobacteria .

What are the optimal storage and handling conditions for recombinant A. marina NAD(P)H-quinone oxidoreductase subunit L?

Based on manufacturer recommendations for the recombinant protein, the following storage and handling protocols should be maintained :

• Storage temperature:

  • Primary storage at -20°C

  • For extended storage periods, -80°C is recommended

  • Avoid repeated freezing and thawing cycles

• Buffer composition:

  • Tris-based buffer with 50% glycerol, specifically optimized for this protein

  • The high glycerol content provides stability during freeze-thaw cycles

• Working solutions:

  • Store working aliquots at 4°C for up to one week

  • Prepare small aliquots to minimize freeze-thaw cycles

• Handling precautions:

  • Due to the membrane-associated nature of this protein, avoid conditions that promote aggregation

  • Maintain reducing conditions when appropriate to preserve any reactive cysteine residues

  • Consider the addition of mild detergents for assays requiring solubilized protein

These handling conditions are critical for maintaining the structural integrity and enzymatic activity of the protein, particularly given its likely membrane association in vivo.

What expression systems have proven most effective for producing recombinant A. marina NAD(P)H-quinone oxidoreductase subunit L?

While the search results don't specify the expression system used for the commercially available recombinant protein, general considerations for membrane-associated proteins from cyanobacteria suggest the following expression systems would be most appropriate:

• E. coli-based systems:

  • BL21(DE3) strains with modifications to enhance membrane protein expression

  • C41(DE3) and C43(DE3) strains specifically engineered for membrane protein expression

  • Co-expression with chaperones to enhance proper folding

• Expression tags and fusion partners:

  • The commercial product notes "The tag type will be determined during production process" , suggesting flexibility based on expression optimization

  • Common options include His6-tags for purification and MBP or SUMO tags for solubility enhancement

• Induction conditions:

  • Lower temperatures (16-20°C) during induction

  • Reduced IPTG concentrations for slower, more controlled expression

  • Extended expression times (24-48 hours)

• Extraction considerations:

  • Specialized detergents for membrane protein solubilization

  • Gentle extraction procedures to maintain native conformation

The selection of an appropriate expression system would need to balance protein yield with proper folding and maintenance of enzymatic activity.

How does the structure of NAD(P)H-quinone oxidoreductase subunit L contribute to the unique photosynthetic capabilities of A. marina?

The NAD(P)H-quinone oxidoreductase subunit L likely contributes to A. marina's unique photosynthetic capabilities through specialized structural adaptations that support far-red light utilization. While detailed structural information specific to this subunit is limited in the search results, we can infer its role based on the context of A. marina's distinctive photosynthetic apparatus.

The protein functions within a system that has evolved to utilize chlorophyll d, which absorbs at longer wavelengths than the more common chlorophyll a. This adaptation requires coordinated modifications throughout the photosynthetic electron transport chain, including:

• Membrane positioning: The hydrophobic nature of the amino acid sequence (MVVNLILLLGLLGGYLLVMPAITYFYLQKRWYVASSLERGFMYFLVFFFFPSLLLLSPFLNFRPQPRKI) suggests transmembrane localization that could position the enzyme optimally relative to photosystems adapted for far-red light.

• Redox tuning: The protein likely contains modified redox centers calibrated to the lower energy input from chlorophyll d-based photosystems.

• Complex assembly: As part of the larger NAD(P)H dehydrogenase I (NDH-1) complex, subunit L may facilitate structural arrangements that optimize electron transfer from NAD(P)H to quinones in this specialized photosynthetic system.

What is the proposed catalytic mechanism of NAD(P)H-quinone oxidoreductase in A. marina compared to similar enzymes?

Based on what we know about NAD(P)H quinone oxidoreductases in general, and contextualizing this to A. marina's unique photosynthetic system, the catalytic mechanism likely follows a substituted enzyme (ping-pong) mechanism similar to that described for NQO1 , but with adaptations specific to the cyanobacterial photosynthetic electron transport chain:

• Initial binding: NAD(P)H binds to the enzyme's active site, which contains a tightly bound FAD cofactor.

• First half-reaction: NAD(P)H transfers a hydride to the FAD, reducing it to FADH₂. The oxidized NAD(P)+ then dissociates from the active site.

• Second substrate binding: A quinone substrate enters the active site.

• Second half-reaction: The reduced FADH₂ transfers electrons to the quinone, reducing it to quinol. This is typically a two-electron reduction that avoids the formation of reactive semiquinone intermediates.

• Product release: The reduced quinol dissociates, completing the catalytic cycle.

In A. marina specifically, this mechanism must be tuned to the energetics of its chlorophyll d-based photosynthetic system. The enzyme may have evolved specific adaptations to maintain efficient electron transfer despite the ~80 mV lower energy input available from its far-red light-driven photosystems . These adaptations could include modified redox potentials of the FAD cofactor or structural changes in the quinone binding site to optimize interaction with quinones specific to A. marina's thylakoid membranes.

How does A. marina NAD(P)H-quinone oxidoreductase compare to homologous proteins in other cyanobacteria?

Comparative analysis of A. marina NAD(P)H-quinone oxidoreductase with homologous proteins in other cyanobacteria reveals important evolutionary adaptations related to its unique photosynthetic capabilities:

The NAD(P)H-quinone oxidoreductase in A. marina likely contains specific amino acid substitutions that optimize its function within this modified photosynthetic system. While maintaining the core catalytic mechanism, these adaptations would ensure efficient electron transport despite the lower energy input from chlorophyll d photosystems. This represents a remarkable example of how electron transport components can be fine-tuned through evolution to accommodate major shifts in light-harvesting strategy while preserving the fundamental chemistry of photosynthesis.

What evolutionary insights can be gained from studying the genomic context of the ndhL gene in A. marina?

The genomic context of the ndhL gene (AM1_1349) in A. marina provides valuable evolutionary insights into niche adaptation and genome expansion in cyanobacteria:

• Genomic expansion: The A. marina genome, at 8.3 million base pairs, is among the largest bacterial genomes sequenced, with nine single-copy plasmids containing over 25% of the putative ORFs . This expansion suggests extensive gene acquisition events that may have facilitated its adaptation to specialized ecological niches.

• Gene duplication patterns: The genome shows heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements , which could account for genetic mobility and genome expansion. This potentially provided the genetic plasticity necessary for evolving novel photosynthetic capabilities.

• Acquisition of specialized pathways: The genomic context may reveal horizontal gene transfer events that contributed to A. marina's unique capabilities, including its unusual complement of genes for phycobiliproteins and antenna proteins that differ from those in other cyanobacteria like Prochlorococcus .

• Radical SAM enzymes: A. marina codes for 12 proteins with putative radical SAM motifs, far more than expected from an oxygen-producing cyanobacterium . Two of these (AM1_5023 and AM1_5798) share very little homology with other sequenced cyanobacteria, suggesting unique enzymatic capabilities potentially related to its specialized metabolism.

• Phylogenetic anomalies: Some genes in A. marina show unusual phylogenetic placement, such as the divinyl chlorophyllide reductase (AM1_2394), which branches near alphaproteobacteria while A. marina also contains a normal copy of the cyanobacterial-type divinyl reductase . This suggests complex evolutionary history involving potential horizontal gene transfer events.

These genomic features collectively establish Acaryochloris as "a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria" .

What are the typical difficulties encountered when working with recombinant NAD(P)H-quinone oxidoreductase, and how can they be addressed?

Researchers working with recombinant NAD(P)H-quinone oxidoreductase subunit L from A. marina may encounter several challenges. Here are common issues and recommended solutions:

• Protein solubility issues:

  • Challenge: The protein's hydrophobic nature suggests membrane association, which may cause aggregation in aqueous solutions.

  • Solution: Use mild detergents (DDM, CHAPS) at concentrations just above their critical micelle concentration. Alternatively, consider nanodisc technology to provide a membrane-like environment.

• Maintaining enzymatic activity:

  • Challenge: Loss of cofactors or structural integrity during purification.

  • Solution: Supplement purification buffers with FAD, maintain reducing conditions with dithiothreitol or β-mercaptoethanol, and consider the addition of glycerol (10-20%) to stabilize the protein structure.

• Assay interference:

  • Challenge: NAD(P)H absorption overlap with other assay components.

  • Solution: Consider fluorescence-based assays or coupled enzyme systems that can monitor activity through secondary reactions with distinct spectral properties.

• Storage stability:

  • Challenge: Activity loss during storage.

  • Solution: Follow the recommended storage conditions: primary storage at -20°C, extended storage at -80°C, and working aliquots at 4°C for up to one week . Avoid repeated freeze-thaw cycles.

• Reconstitution into physiologically relevant systems:

  • Challenge: Recreating the native membrane environment.

  • Solution: Consider proteoliposome reconstitution with lipid compositions that mimic A. marina thylakoid membranes, possibly including specialized pigments like chlorophyll d.

• Heterologous expression yield:

  • Challenge: Low expression levels in common expression systems.

  • Solution: Optimize codon usage for the expression host, consider specialized strains for membrane protein expression, and explore fusion partners that enhance expression while allowing for their subsequent removal.

How can researchers validate the functional integrity of purified recombinant A. marina NAD(P)H-quinone oxidoreductase?

Validating the functional integrity of purified recombinant A. marina NAD(P)H-quinone oxidoreductase requires a multi-faceted approach:

• Spectroscopic characterization:

  • UV-visible absorption spectroscopy to confirm the presence of bound FAD cofactor (typical absorption peaks at approximately 375 and 450 nm)

  • Fluorescence spectroscopy to assess FAD binding (excitation at 450 nm, emission at 525 nm)

  • Circular dichroism to evaluate secondary structure integrity

• Functional assays:

  • NAD(P)H oxidation assays monitoring decrease in absorbance at 340 nm

  • Quinone reduction assays using various quinone substrates

  • Inhibition studies with known inhibitors of quinone oxidoreductases (e.g., dicoumarol)

  • Comparison of activity with different electron acceptors to establish substrate specificity profile

• Structural integrity assessment:

  • Size-exclusion chromatography to confirm oligomeric state (likely homodimeric based on other quinone oxidoreductases)

  • Dynamic light scattering to assess homogeneity and detect aggregation

  • Thermal shift assays to determine stability under various conditions

• Comparative metrics:

  • Establishing baseline kinetic parameters (Km, kcat) for comparison with published values for similar enzymes

  • Activity comparisons at different pH values and temperatures to establish optimal conditions

  • Assessing activity in the presence of various ions and potential regulatory molecules

A comprehensive validation would include measuring specific activity (units/mg protein) under standardized conditions that can serve as a reference for future preparations and experiments.

What are the most promising research avenues for understanding the role of NAD(P)H-quinone oxidoreductase in far-red light photosynthesis?

Several promising research avenues could advance our understanding of NAD(P)H-quinone oxidoreductase's role in far-red light photosynthesis:

• Structure-function studies: Determining the high-resolution structure of the entire NAD(P)H dehydrogenase I complex from A. marina would provide crucial insights into adaptations for far-red light photosynthesis. Recent advances in cryo-electron microscopy, similar to those used for A. marina's PSI structure , could be applied to this complex.

• Redox tuning mechanisms: Investigating how the redox potentials of electron transfer components in A. marina have been tuned to accommodate the ~80 mV lower energy input from chlorophyll d-based photosystems . This could involve electrochemical studies, protein engineering, and comparative analysis across species.

• Synthetic biology applications: Exploring the potential to engineer far-red light photosynthetic capabilities into other organisms by transferring components of A. marina's electron transport chain, including modified NAD(P)H-quinone oxidoreductase complexes.

• Environmental adaptation studies: Examining how A. marina's unique electron transport components, including NAD(P)H-quinone oxidoreductase, contribute to its ecological niche adaptation in environments dominated by far-red light.

• Integration with artificial photosynthetic systems: Investigating how the principles of A. marina's electron transport chain could inform the development of artificial photosynthetic systems capable of utilizing the full solar spectrum, including far-red wavelengths.

• Comparative genomics with other far-red light phototrophs: Expanding comparative studies to include recently discovered far-red light utilizing organisms to identify convergent evolutionary solutions to the challenges of low-energy photosynthesis.

These research directions could not only advance our understanding of photosynthetic diversity but also contribute to applications in renewable energy, agriculture, and biotechnology.

How might understanding A. marina NAD(P)H-quinone oxidoreductase contribute to synthetic biology applications?

Understanding A. marina's NAD(P)H-quinone oxidoreductase could enable several innovative synthetic biology applications:

• Extended spectrum photosynthesis: Engineering crop plants or algae to incorporate components of A. marina's electron transport chain could potentially extend their photosynthetic range into the far-red spectrum, increasing light utilization efficiency and potentially crop yields. The NAD(P)H-quinone oxidoreductase components would need to be optimized to interface with existing photosynthetic machinery.

• Bioremediation applications: Enzymes like NAD(P)H-quinone oxidoreductase catalyze the reduction of various organic compounds including potential environmental pollutants . Engineering variants based on A. marina's enzyme could yield specialized bioremediation tools for specific contaminants.

• Biofuel production: The ability to harvest far-red light could enhance biofuel production in engineered organisms by utilizing wavelengths that penetrate deeper into high-density cultures, potentially increasing productivity.

• Novel bioelectronic interfaces: A. marina's adaptations for efficient electron transport despite lower energy input could inform the design of biological-electronic interfaces in applications such as microbial fuel cells or biosensors that operate under energy-limited conditions.

• Protein engineering platforms: The study of how A. marina has fine-tuned its electron transport components could provide valuable principles for protein engineering, particularly for optimizing enzymes to function under non-standard conditions.

• Photobioreactors with expanded light utilization: Engineered microorganisms incorporating A. marina's photosynthetic components could be used in specialized photobioreactors designed to utilize both visible and far-red light, potentially increasing production efficiency.