Recombinant Synechococcus sp. NAD (P)H-quinone oxidoreductase subunit L

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Description

Functional Role in Cyanobacterial Metabolism

NDH-1 in Synechococcus sp. is integral to energy metabolism, with subunit L contributing to:

  • Electron Transport: Mediates proton-pumping activity by transferring electrons from NAD(P)H to plastoquinone, generating a proton gradient for ATP synthesis .

  • Metabolic Regulation: Overexpression of ndhL in engineered strains enhances NADPH and ATP production, supporting pathways like glycolysis and amino acid synthesis .

  • Stress Response: Upregulates malonyl-CoA reductase (MCR) activity, linking it to 3-hydroxypropionate (3-HP) biosynthesis in metabolic engineering studies .

Proteomic Findings

  • Strains overexpressing ndhL show increased levels of enzymes in oxidative phosphorylation (e.g., Slr0844, annotated as NAD(P)H-quinone oxidoreductase subunit F) .

  • Metabolomic profiling reveals elevated NADPH/NADP⁺ ratios and ATP, suggesting enhanced photosynthetic efficiency .

Metabolic Engineering

  • 3-HP Biosynthesis: Overexpression of ndhL in Synechocystis enhances malonyl-CoA flux, a precursor for 3-HP, by upregulating ACP-S-malonyl transferase (Slr2023) .

  • Carbon/Nitrogen Metabolism: Modulates α-ketoglutarate (AKG) levels, balancing TCA cycle intermediates and amino acid synthesis .

Conformational Dynamics

Comparative studies of NAD(P)H-quinone oxidoreductases (e.g., human NQO1) reveal:

  • A conserved ping-pong mechanism, where cofactor (NAD(P)H) and substrate (quinone) binding induce conformational changes in loops L5 and L9, regulating catalytic site accessibility .

  • Structural plasticity enables accommodation of diverse substrates, from benzoquinones to chemotherapeutic agents .

Superfamily Relationships

NDH-1 shares evolutionary ties with azoreductases and flavin-dependent oxidoreductases, evidenced by structural overlaps in FMN-binding domains .

Oxidative Stress Mitigation

  • Recombinant ndhL indirectly modulates reactive oxygen species (ROS) by maintaining NADPH pools, critical for antioxidant pathways .

  • Human NQO1 homologs scavenge superoxide radicals via NADH-dependent mechanisms, suggesting analogous roles in cyanobacteria .

Industrial Relevance

  • Biofuel Production: Enhanced NADPH supply supports fatty acid biosynthesis in engineered cyanobacteria .

  • Pharmaceuticals: NDH-1’s redox-switching capability is exploitable for activating prodrugs in cancer therapy .

Product Specs

Form
Lyophilized powder
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your needs whenever possible.
Lead Time
Delivery time may vary depending on the purchase method and location. Please consult your local distributor for specific delivery time estimates.
Note: All proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please communicate with us in advance. Additional fees will apply.
Notes
Repeated freezing and thawing is not recommended. For optimal results, store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly prior to opening to ensure the contents are settled at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, we recommend adding 5-50% glycerol (final concentration) and aliquoting the solution at -20°C/-80°C. Our default final glycerol concentration is 50%, which can serve as a reference point.
Shelf Life
Shelf life is influenced by various factors, including storage conditions, buffer components, storage temperature, and the inherent stability of the protein itself.
Generally, the shelf life for the liquid form is 6 months at -20°C/-80°C. For the lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is essential for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
The tag type is determined during production. If you have specific tag type requirements, please inform us, and we will prioritize developing the specified tag.
Synonyms
ndhL; SynWH7803_0730; NAD(PH-quinone oxidoreductase subunit L; NAD(PH dehydrogenase I subunit L; NDH-1 subunit L; NDH-L
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-81
Protein Length
full length protein
Species
Synechococcus sp. (strain WH7803)
Target Names
ndhL
Target Protein Sequence
METLLNAIPQETLLVIGAYGALGAAYLVVIPLFLYFWMNRRWTVMGKLERLGIYGLVFLF FPGLILFAPFLNLRMSGQGDV
Uniprot No.

Target Background

Function
NDH-1 facilitates electron transfer from an unknown electron donor, through FMN and iron-sulfur (Fe-S) centers, to quinones in the respiratory and/or photosynthetic chain. In this species, the immediate electron acceptor for the enzyme is believed to be plastoquinone. The enzyme couples the redox reaction with proton translocation, thereby conserving redox energy in a proton gradient. Cyanobacterial NDH-1 also plays a role in inorganic carbon concentration.
Database Links
Protein Families
Complex I NdhL subunit family
Subcellular Location
Cellular thylakoid membrane; Multi-pass membrane protein.

Q&A

What is the fundamental role of NAD(P)H dehydrogenases in Synechococcus sp.?

NAD(P)H dehydrogenases in Synechococcus sp. function primarily in transferring electrons derived from organic molecules into the plastoquinone (PQ) pool. These enzymes fall into two distinct classes: type 1 NAD(P)H dehydrogenases (NDH-1) and type 2 NAD(P)H dehydrogenases (NDH-2). The NDH-1 enzymes are proton-pumping multisubunit complexes that resemble mitochondrial complex I, residing predominantly in the thylakoid membrane where they participate in cyclic electron transfer (CET), respiration, and carbon uptake . In contrast, NDH-2 enzymes consist of single polypeptides with molecular masses of approximately 50 kD and do not contribute to generating the proton gradient across the membrane .

While NDH-1 complexes contain multiple subunits including subunit L, the NDH-2 family provides additional electron transfer pathways that may serve as redox sensors responding to the redox state of the PQ pool, rather than having significant catalytic roles in respiration . In organisms where NADH oxidation is carried out solely by NDH-2s, these enzymes primarily perform respiratory chain-linked NADH turnover and subsequent electron donation to protein complexes that produce the proton gradient powering ATP production .

How do NAD(P)H dehydrogenases contribute to redox balance in cyanobacteria?

NAD(P)H dehydrogenases play a crucial role in maintaining redox homeostasis in cyanobacteria by regulating the NAD+/NADH balance. Research with Synechocystis sp. PCC 6803 demonstrated that when all three NDH-2 enzymes (NdbA, NdbB, and NdbC) were deleted, reduced NADH strongly prevailed, indicating these enzymes' importance in maintaining proper redox balance . This regulation affects multiple cellular processes, including carbon allocation between storage and biosynthesis pathways, and even influences cell division .

The function of these enzymes extends beyond simple electron transfer. For instance, in Synechococcus sp. PCC7002, the enzymatic activity of sulfide:quinone oxidoreductase (SQR), which shares electron transfer pathways with NAD(P)H dehydrogenases, affects the expression of key photosynthetic genes. Studies have shown that deletion of the sqr gene increases the expression of genes involved in photosynthesis, including psbA1, psbA2, and psbA3 (encoding D1 protein isoforms), as well as rbcX, rbcL, and rbcS (encoding RuBisCO), and tkt (encoding transketolase) . This indicates a regulatory connection between electron transfer enzymes and photosynthetic machinery.

What distinguishes subunit L from other components of the NAD(P)H-quinone oxidoreductase complex?

Subunit L is a key component of the type 1 NAD(P)H dehydrogenase (NDH-1) complex, which differs substantially from the single-polypeptide NDH-2 enzymes. The NDH-1 complex in cyanobacteria resembles the mitochondrial complex I but has evolved specific adaptations for photosynthetic organisms. Subunit L contributes to the core structure of this multisubunit complex and participates in proton translocation across the thylakoid membrane, a function absent in NDH-2 enzymes .

While NDH-2 enzymes can be characterized by their relatively simple structure and specific binding motifs for cofactors like FAD and substrates like NAD(P)H, the NDH-1 complex's subunit L operates within a more intricate protein assembly. The functional importance of subunit L must be understood in the context of the entire NDH-1 complex, which is primarily involved in cyclic electron flow around photosystem I, carbon dioxide concentration mechanisms, and respiratory electron transport in cyanobacteria.

How does the genetic modification of NAD(P)H dehydrogenase genes affect sulfide metabolism in Synechococcus sp.?

Genetic modification of NAD(P)H dehydrogenase genes has revealed significant impacts on sulfide metabolism in Synechococcus sp. PCC7002. Research has shown that the deletion of the sqr gene, which encodes sulfide:quinone oxidoreductase (an enzyme that participates in electron transfer pathways alongside NAD(P)H dehydrogenases), results in decreased cellular S0 levels and altered expression of photosynthetic genes . The Δsqr mutant exhibited increased expression of key genes involved in photosynthesis but was less competitive than the wild type in cocultures, suggesting that proper sulfide metabolism provides growth advantages .

The relationship between NAD(P)H dehydrogenases and sulfide metabolism involves complex electron transfer pathways. When the sqr gene is intact, Synechococcus sp. PCC7002 can oxidize self-produced sulfide to S0, present as persulfide and polysulfide in the cell . This oxidation process helps maintain cellular sulfane sulfur levels, which appears to be advantageous for growth. Additionally, the presence of SQR and persulfide dioxygenase (PDO) allows Synechococcus sp. PCC7002 to oxidize exogenous sulfide, enabling tolerance to high sulfide levels that would otherwise be toxic . This detoxification capability may provide an ecological advantage in oxygen minimum zones (OMZs) where sulfide can accumulate to high concentrations .

What are the molecular mechanisms underlying the differential expression of NAD(P)H dehydrogenase isoforms under varying environmental conditions?

The molecular mechanisms governing the differential expression of NAD(P)H dehydrogenase isoforms in Synechococcus sp. involve complex regulatory networks responsive to environmental cues. Under sulfide stress conditions, expression patterns change significantly. For instance, in the presence of elevated sulfide or sulfane sulfur, specific stress response systems are activated. Research has shown that all six peroxiredoxins in Synechococcus sp. PCC7002 are induced by elemental sulfur (S8), suggesting a coordinated response to sulfur stress .

The regulatory mechanisms likely involve redox-sensitive transcription factors that can sense changes in cellular redox status. For example, in Synechocystis sp. PCC 6803, it has been suggested that NDH-2 enzymes function as redox sensors responding to the redox state of the plastoquinone pool . This sensing ability could facilitate appropriate gene expression changes in response to environmental fluctuations.

The differential expression may also be influenced by ecological factors. Approximately 45% of sequenced cyanobacterial genomes contain SQR, compared to only 21% of sequenced bacterial genomes generally, indicating the particular importance of sulfide-related electron transfer mechanisms in cyanobacteria . This widespread distribution suggests that environmental selective pressures, particularly in sulfide-rich habitats like OMZs, have shaped the evolution and regulation of these electron transfer enzymes in cyanobacteria.

How do recombinant NAD(P)H-quinone oxidoreductases compare with native enzymes in terms of substrate specificity and catalytic efficiency?

In functional complementation studies, certain NDH-2 enzymes have demonstrated cross-species activity. For instance, NdbB from Synechocystis and NDA2 from C. reinhardtii could functionally complement an Escherichia coli mutant lacking both NDH-1 and NDH-2s . This suggests conservation of core catalytic functions despite variations in protein sequence and cellular context.

For subunit L specifically, recombinant expression presents challenges due to its integration within the larger NDH-1 complex. Successful recombinant production often requires co-expression with other subunits or careful design of soluble derivatives. The substrate specificity of the reconstituted complex may differ from the native complex due to subtle changes in protein-protein interactions that influence electron transfer pathways.

What are the optimal expression systems and purification strategies for obtaining active recombinant NAD(P)H-quinone oxidoreductase subunit L?

The expression and purification of active recombinant NAD(P)H-quinone oxidoreductase subunit L requires careful consideration of several factors to maintain the protein's native structure and function. Based on successful approaches with similar proteins, the following strategies are recommended:

Expression Systems:

  • E. coli BL21(DE3) with pET-based vectors provides a robust platform for initial expression trials, particularly when the target protein is tagged with a solubility enhancer like SUMO or MBP

  • For proteins requiring post-translational modifications, yeast systems (Pichia pastoris or Saccharomyces cerevisiae) may yield better results

  • For maintaining proper membrane association, insect cell expression systems using baculovirus vectors can preserve the native folding environment

Purification Strategies:

  • Two-step purification using affinity chromatography (Ni-NTA for His-tagged constructs) followed by size exclusion chromatography

  • Inclusion of appropriate detergents (such as 0.02% DDM or 0.5% CHAPS) during extraction and purification if the protein has membrane-associated domains

  • Maintaining reducing conditions throughout purification by including 1-5 mM DTT or 2-mercaptoethanol to prevent oxidation of critical cysteine residues

Activity preservation often requires reconstitution with appropriate cofactors (FAD or FMN) and testing with various electron acceptors such as decylubiquinone or menadione. Functional validation can be performed through spectrophotometric assays monitoring NAD(P)H oxidation at 340 nm or reduction of artificial electron acceptors.

How can researchers effectively analyze the interaction between NAD(P)H dehydrogenases and sulfide metabolism pathways?

Analyzing interactions between NAD(P)H dehydrogenases and sulfide metabolism pathways requires an integrated approach combining genetic, biochemical, and physiological methods. Based on successful research strategies documented in the literature, the following methodological approach is recommended:

Genetic Approaches:

  • Generate specific gene knockouts (e.g., Δsqr mutants) to disrupt key components of the pathway

  • Create complementation strains to verify phenotype restoration

  • Implement inducible expression systems to control protein levels during experiments

Biochemical Analyses:

  • Measure cellular sulfane sulfur levels using specific probes like SSP4 (sulfane sulfur probe 4)

  • Quantify sulfide production/consumption rates using colorimetric assays with N,N-dimethyl-p-phenylenediamine

  • Assess enzyme activities in cell extracts or with purified recombinant proteins using spectrophotometric methods

Physiological Measurements:

  • Monitor growth rates in mixed cultures to assess competitive fitness

  • Evaluate tolerance to exogenous sulfide at varying concentrations

  • Measure photosynthetic efficiency using PAM fluorometry to detect changes in electron transport

For studying the specific role of subunit L, researchers should consider co-immunoprecipitation experiments to identify interaction partners and blue native PAGE to analyze complex assembly. Transcriptomic and proteomic approaches can provide comprehensive insights into how disruption of these pathways affects broader cellular processes. Recent studies have demonstrated the value of competition experiments in mixed cultures for assessing the ecological significance of these pathways .

What advanced spectroscopic techniques are most informative for studying electron transfer in NAD(P)H-quinone oxidoreductase complexes?

Advanced spectroscopic techniques provide crucial insights into the electron transfer mechanisms within NAD(P)H-quinone oxidoreductase complexes. The following methods have proven particularly valuable for studying these systems:

Time-Resolved Spectroscopy:

  • Ultrafast transient absorption spectroscopy can track electron transfer events on the picosecond to microsecond timescale

  • Fluorescence lifetime measurements help identify changes in cofactor environments during catalysis

  • Stopped-flow spectroscopy allows monitoring of reaction kinetics with millisecond resolution

Electron Paramagnetic Resonance (EPR):

  • Continuous wave EPR detects paramagnetic species including flavin semiquinones and iron-sulfur clusters

  • Pulse EPR techniques like HYSCORE provide information about the molecular environment of paramagnetic centers

  • Spin labeling approaches combined with DEER spectroscopy can measure distances between specific protein domains

Resonance Raman Spectroscopy:

  • Provides vibrational information about cofactors (FAD, FMN, quinones) during catalysis

  • Can be performed with different excitation wavelengths to selectively probe specific chromophores

  • Time-resolved measurements capture transient intermediates during electron transfer

For comprehensive analysis, these spectroscopic approaches should be combined with structural methods like X-ray crystallography or cryo-EM. Recent advances in native mass spectrometry also offer insights into complex assembly and stability. When studying recombinant subunit L specifically, reconstitution with other complex components may be necessary to observe physiologically relevant electron transfer events.

How can understanding NAD(P)H dehydrogenase function in Synechococcus sp. contribute to bioremediation of sulfide-rich environments?

Understanding NAD(P)H dehydrogenase function in Synechococcus sp. offers significant potential for bioremediation applications in sulfide-rich environments. Research has demonstrated that Synechococcus sp. PCC7002 possesses robust mechanisms for sulfide detoxification, primarily through the action of sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) . These enzymes collectively oxidize sulfide to sulfite and thiosulfate, resembling pathways recently reported in heterotrophic bacteria .

The detoxification capability enables Synechococcus sp. PCC7002 to survive better than SQR-deficient mutants in environments with high sulfide concentrations . This natural mechanism could be enhanced through genetic engineering to develop strains with improved sulfide oxidation capacity for bioremediation purposes. The widespread distribution of SQR in cyanobacteria (approximately 45% of sequenced cyanobacterial genomes contain SQR, compared to only 21% of sequenced bacterial genomes generally) indicates evolutionary adaptation to sulfide-rich environments .

From a methodological perspective, researchers should consider:

  • Evaluating sulfide tolerance thresholds of wild-type vs. engineered strains

  • Measuring sulfide oxidation rates under various environmental conditions

  • Assessing the impact of enhanced sulfide metabolism on photosynthetic oxygen production

  • Developing immobilization techniques for deploying cyanobacteria in field applications

Additionally, the oxygen produced through photosynthesis by these organisms could help relieve deoxygenation in oxygen minimum zones (OMZs), potentially creating a dual remediation effect: detoxifying sulfide while increasing oxygen levels .

What insights from NAD(P)H dehydrogenase research in cyanobacteria can be applied to improving photosynthetic efficiency in other organisms?

Research on NAD(P)H dehydrogenases in cyanobacteria has revealed several key insights with potential applications for improving photosynthetic efficiency in other organisms. The study of Synechococcus sp. PCC7002 has demonstrated complex interplays between electron transfer pathways, sulfur metabolism, and photosynthetic performance that could inform genetic engineering strategies.

One significant finding is that the deletion of the sqr gene in Synechococcus sp. PCC7002 leads to increased expression of key photosynthetic genes, including those encoding D1 protein isoforms (psbA1, psbA2, and psbA3), RuBisCO components (rbcX, rbcL, and rbcS), and transketolase (tkt) of the Calvin-Benson-Bassham cycle . This upregulation suggests that modifying electron transfer pathways can influence photosynthetic gene expression, potentially offering a strategy for enhancing carbon fixation.

The research also highlights the importance of redox balance in optimizing photosynthetic performance. In Synechocystis sp. PCC 6803, NDH-2 enzymes appear to function as redox sensors responding to the redox state of the plastoquinone pool . This sensing capability could be engineered into other photosynthetic organisms to improve their ability to adapt to changing light conditions.

From a methodological perspective, researchers exploring these applications should:

  • Employ comparative genomics to identify conserved components across diverse photosynthetic organisms

  • Utilize targeted genetic engineering to modify specific electron transfer components

  • Implement high-throughput phenotyping to assess impacts on growth and photosynthetic parameters

  • Develop mathematical models of electron flow to predict the effects of genetic modifications

How might recombinant NAD(P)H dehydrogenases be utilized in biosensor development for environmental monitoring?

Recombinant NAD(P)H dehydrogenases offer promising potential for biosensor development, particularly for monitoring environmental sulfide levels and redox conditions. These enzymes possess several characteristics that make them suitable for biosensor applications:

Sensing Capabilities:

  • NDH-2 enzymes in cyanobacteria like Synechocystis appear to function as redox sensors responding to the redox state of the plastoquinone pool

  • SQR enzymes, which share electron transfer pathways with NAD(P)H dehydrogenases, demonstrate high sensitivity to sulfide and can operate across a range of concentrations

  • Peroxiredoxins induced under sulfur stress conditions could serve as indicators for sulfane sulfur species

Methodological Approaches for Biosensor Development:

  • Engineer chimeric proteins combining the sensing domains of these enzymes with reporter elements (fluorescent proteins, electrochemical mediators)

  • Develop whole-cell biosensors using genetically modified cyanobacteria with reporter genes under the control of promoters responsive to sulfide or redox stress

  • Create immobilized enzyme electrodes using purified recombinant proteins for direct electrochemical detection

  • Implement microfluidic platforms for high-throughput screening of environmental samples

The specificity of these enzymes for particular substrates enables selective detection of target compounds. For instance, SQR's specificity for sulfide could form the basis for highly selective sulfide sensors for monitoring aquatic environments, particularly in oxygen minimum zones where sulfide accumulation presents ecological hazards . Similarly, the differential substrate preferences of various NAD(P)H dehydrogenase isoforms could be exploited to develop sensors for specific electron donors or acceptors in environmental or clinical samples.

What are the key challenges in studying structure-function relationships of NAD(P)H-quinone oxidoreductase subunit L in cyanobacteria?

Studying the structure-function relationships of NAD(P)H-quinone oxidoreductase subunit L in cyanobacteria presents several significant challenges that researchers must address through innovative methodological approaches:

Structural Complexity Challenges:

  • Subunit L functions as part of a large multiprotein complex, making it difficult to study in isolation

  • The membrane-associated nature of the NDH-1 complex complicates structural determination using traditional crystallography

  • The dynamic interactions between subunits during electron transfer are difficult to capture in static structural models

Functional Analysis Challenges:

  • Distinguishing the specific role of subunit L within the larger complex requires precise mutagenesis strategies

  • The redundancy of some electron transfer pathways can mask phenotypes in single-subunit knockout studies

  • The potential dual functions in cyclic electron flow and respiration create complex phenotypes that are challenging to interpret

To address these challenges, researchers should consider employing:

  • Cryo-electron microscopy to determine structures of intact complexes in different functional states

  • Site-directed mutagenesis targeting conserved residues identified through comparative genomics

  • Time-resolved spectroscopic techniques to track electron transfer through the complex

  • Reconstitution studies with defined subunit compositions to determine minimal functional units

  • Advanced imaging techniques like super-resolution microscopy to visualize complex assembly in vivo

Understanding these structure-function relationships is crucial for developing strategies to enhance photosynthetic efficiency or engineer novel electron transfer pathways for biotechnological applications.

How might climate change impact the ecological significance of sulfide detoxification pathways in marine cyanobacteria?

Climate change is expected to significantly alter the ecological significance of sulfide detoxification pathways in marine cyanobacteria like Synechococcus sp. through multiple interconnected mechanisms:

Expanding Oxygen Minimum Zones (OMZs):

  • Rising ocean temperatures, decreased oxygen solubility, and increased biological activities (including photosynthesis and respiration) are leading to the expansion of OMZs

  • Eutrophication from excessive nutrient input from agriculture and aquaculture contributes to coastal water deoxygenation

  • These expanding OMZs may experience sporadic accumulation of sulfide, presenting increased toxicity challenges for marine organisms

Increased Selective Pressure:

  • Cyanobacteria possessing efficient sulfide detoxification mechanisms, such as the SQR-PDO pathway in Synechococcus sp. PCC7002, will likely have competitive advantages in these changing environments

  • The wide distribution of SQR in cyanobacteria (approximately 45% of sequenced genomes) suggests evolutionary adaptation to sulfide exposure that may become increasingly important

Methodologically, researchers investigating these ecological shifts should:

  • Conduct competition experiments between wild-type and sulfide detoxification-deficient strains under projected future ocean conditions

  • Perform metagenomic analyses of OMZ communities to track changes in the prevalence of sulfide metabolism genes

  • Develop models integrating sulfur and carbon cycles to predict ecosystem-level responses to climate change

  • Implement mesocosm experiments simulating future ocean conditions to observe community-level adaptations

Understanding these ecological dynamics will be crucial for predicting how marine primary production may change in response to climate-driven expansion of OMZs and associated sulfide accumulation.

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