Recombinant Bacillus cereus Ferredoxin--NADP reductase 2 (BCE_5065)

What is Ferredoxin--NADP reductase 2 (BCE_5065) and what protein family does it belong to?

Ferredoxin--NADP reductase 2 (BCE_5065) is a 331-amino acid protein with a molecular mass of approximately 36.7 kDa derived from Bacillus cereus strain ATCC 10987/NRS 248 . This enzyme belongs specifically to the ferredoxin--NADP reductase type 2 family, a group of oxidoreductases involved in electron transfer processes that couple the oxidation of ferredoxin to the reduction of NADP+ . The protein contains the characteristic domains and conserved residues essential for binding both the ferredoxin substrate and the NADP+ cofactor, enabling efficient electron transfer. BCE_5065 represents one of several oxidoreductases encoded in the B. cereus genome, highlighting the complexity of the organism's redox regulation systems. The complete amino acid sequence of BCE_5065 has been determined, providing a foundation for structural studies and functional analyses .

How does BCE_5065 structurally compare with other ferredoxin reductases in Bacillus cereus?

BCE_5065 (Ferredoxin--NADP reductase 2) exhibits structural properties that distinguish it from other reductases in B. cereus, including the related Ferredoxin--NADP reductase 1. When examining the structural organization, BCE_5065 contains the characteristic NADPH-binding and FAD-binding domains commonly observed in type 2 reductases, but with distinct spatial arrangements that impact substrate specificity . Unlike thioredoxin reductase (TrxR) in B. cereus, BCE_5065 lacks the CXXC catalytic motif that is essential for disulfide reductase activity, which explains its functional divergence from TrxR . Structural analyses of related B. cereus reductases have revealed that TrxR exhibits an asymmetric binding pattern, where only one of two binding sites in the homodimer is occupied with NADPH, suggesting potential half-site reactivity . This characteristic may or may not be shared with BCE_5065, though the structural homology between these enzymes suggests similar binding mechanics may be possible. Comparative analysis with FNR2, which has been established as an endogenous redox partner in the activation of NrdI in class Ib RNR from B. cereus, reveals specific structural adaptations in BCE_5065 that influence its distinctive substrate preferences and catalytic properties .

What is known about the active site architecture of BCE_5065?

The active site of BCE_5065 contains specific structural elements that are critical for its function as a ferredoxin--NADP reductase. Based on analysis of homologous proteins in the ferredoxin--NADP reductase type 2 family, the active site likely includes a binding pocket for FAD, which serves as the initial electron acceptor in the reaction mechanism . The NADPH binding domain appears to contain conserved residues that interact with the nicotinamide moiety and the 2'-phosphate group of NADPH, providing specificity for this cofactor over NAD+ . Critical residues that facilitate electron transfer between FAD and NADP+ are positioned to optimize orbital overlap and electron tunneling efficiency. When comparing to related TrxR-like FNRs in B. cereus, the BCE_5065 active site likely lacks the CXXC catalytic motif found in true thioredoxin reductases, which explains its functional specialization toward ferredoxin rather than thioredoxin substrates . Subtle variations in the electrostatic surface and hydrophobic packing within the active site contribute to differential recognition of ferredoxin versus other potential electron donors, allowing BCE_5065 to fulfill its specific role in the electron transfer networks of B. cereus.

What is the primary biochemical function of BCE_5065 in Bacillus cereus metabolism?

The primary function of BCE_5065 (Ferredoxin--NADP reductase 2) in B. cereus metabolism involves catalyzing the electron transfer from reduced ferredoxin to NADP+, generating NADPH for various anabolic pathways . This reaction is fundamental to maintaining the cellular redox balance and providing reducing power for biosynthetic processes, including fatty acid synthesis, amino acid production, and nucleotide metabolism. Within the B. cereus metabolic network, BCE_5065 likely functions as part of an electron transport chain that couples diverse metabolic processes to the generation of reducing equivalents. Drawing comparisons with other characterized FNRs in B. cereus, BCE_5065 may exhibit specificity for particular ferredoxin partners, allowing for precise regulation of electron flow to specific metabolic pathways . Unlike the thioredoxin reductase (TrxR) system that primarily functions in disulfide reduction, BCE_5065 operates in parallel metabolic pathways involving iron-sulfur proteins, contributing to the metabolic versatility that enables B. cereus to adapt to various environmental conditions . The functional significance of BCE_5065 extends beyond basic metabolism, potentially influencing processes such as oxidative stress response and nutrient assimilation.

How does BCE_5065 participate in electron transfer processes compared to other reductases in B. cereus?

BCE_5065 participates in electron transfer processes through a distinctive mechanism compared to other reductases in B. cereus, particularly in terms of substrate specificity and electron flow directionality. While thioredoxin reductase (TrxR) primarily transfers electrons from NADPH to thioredoxins containing disulfide bonds, BCE_5065 specializes in mediating electron transfer between ferredoxins and NADP+ . Research on related TrxR-like FNRs in B. cereus has shown significant differences in catalytic efficiencies and turnover rates among different FNR-Fld/NrdI pairs, with FNR2 demonstrating the highest turnover number in the activation of NrdI in class Ib RNR . BCE_5065, as Ferredoxin--NADP reductase 2, likely exhibits electron transfer properties that are optimized for its specific physiological role. The absence of the CXXC catalytic motif in BCE_5065, which is essential for disulfide reductase activity in TrxR, structurally explains why it cannot effectively reduce thioredoxin substrates . Instead, BCE_5065 contains specific residues that facilitate interaction with iron-sulfur cluster-containing ferredoxins, enabling efficient coupling of ferredoxin oxidation with NADP+ reduction. This functional specialization allows BCE_5065 to contribute to distinct electron transfer pathways within the complex redox network of B. cereus.

What cofactors are required for BCE_5065 activity and how do they influence catalytic efficiency?

BCE_5065 requires specific cofactors for optimal catalytic activity, with FAD serving as the primary prosthetic group that mediates electron transfer between ferredoxin and NADP+ . The FAD molecule binds tightly within the FAD-binding domain of BCE_5065, where it undergoes reduction and oxidation cycles during catalysis. NADP+ acts as the terminal electron acceptor, binding to the NADPH-binding domain of the enzyme in an orientation that facilitates hydride transfer from the reduced FAD . The interaction between these cofactors significantly influences the catalytic efficiency of BCE_5065, with optimal electron transfer requiring precise alignment of the nicotinamide ring of NADP+ with the isoalloxazine ring of FAD. Studies of related reductases in B. cereus have revealed that asymmetric binding of NADPH can occur, suggesting a potential half-of-the-sites reactivity mechanism that may also apply to BCE_5065 . Metal ions such as Mg2+ or Ca2+ may play auxiliary roles in stabilizing the binding of NADP+ and influencing the enzyme's conformational dynamics during catalysis. The binding affinity of BCE_5065 for its cofactors likely affects its response to changes in the cellular redox state, allowing for regulation of its activity based on the availability of NADP+ and the redox state of ferredoxin partners.

What expression systems yield optimal production of recombinant BCE_5065?

For optimal expression of recombinant BCE_5065, Escherichia coli-based expression systems have demonstrated considerable efficacy for ferredoxin--NADP reductases from Bacillus species . When designing expression constructs, researchers should consider incorporating an N-terminal His-tag or alternative affinity tag to facilitate subsequent purification steps while minimizing interference with the enzyme's active site. The pET expression system, particularly pET28a(+) with a T7 promoter, offers tight regulation and high-level expression when induced with IPTG at concentrations between 0.1-1.0 mM. Expression should be conducted at lower temperatures (16-25°C) following induction to enhance proper protein folding and solubility, with extended expression periods of 16-20 hours often yielding better results than shorter, high-temperature inductions. The expression media should be supplemented with riboflavin (10 μM) to ensure adequate FAD incorporation, as this cofactor is critical for proper folding and activity of BCE_5065. For challenging expression scenarios, specialized E. coli strains such as Rosetta(DE3) or Origami(DE3) may address codon bias issues or facilitate disulfide bond formation, respectively. Alternative expression hosts such as Bacillus subtilis might provide advantages for expressing BCE_5065 in certain research contexts, particularly when post-translational modifications specific to Gram-positive bacteria are required.

What purification strategies yield the highest purity and activity of recombinant BCE_5065?

A multi-step purification strategy is recommended to obtain highly pure and active recombinant BCE_5065. Initially, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides effective capture of His-tagged BCE_5065, with elution performed using an imidazole gradient (50-250 mM) in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol to maintain protein stability . Following IMAC, ion exchange chromatography using a Q-Sepharose column effectively separates BCE_5065 from residual contaminants, utilizing a gradient of 0-500 mM NaCl in 20 mM Tris-HCl pH 7.5. For applications requiring exceptionally high purity, size exclusion chromatography serves as a polishing step, using a Superdex 200 column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5% glycerol. Throughout the purification process, including 1 mM DTT or 2 mM β-mercaptoethanol in all buffers helps maintain the redox state of cysteine residues and preserve enzymatic activity. The purified protein should exhibit the characteristic yellow color of flavoproteins, with an absorption spectrum showing peaks at approximately 375 and 450 nm, confirming proper FAD incorporation. Assessment of protein purity at each purification stage using SDS-PAGE should target >95% homogeneity for the final preparation, with expected migration corresponding to the theoretical molecular weight of 36.7 kDa plus any affinity tag contribution .

How can researchers accurately measure the enzymatic activity of BCE_5065 in vitro?

Accurate measurement of BCE_5065 enzymatic activity requires careful consideration of reaction conditions and detection methods. The standard assay for ferredoxin--NADP reductase activity involves spectrophotometric monitoring of NADPH formation at 340 nm (ε = 6,220 M-1 cm-1) in a reaction mixture containing purified BCE_5065, reduced ferredoxin, and NADP+ . A typical reaction buffer consists of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1 mM EDTA, maintained at 25°C. Reduced ferredoxin can be generated in situ using either chemical reducing agents like sodium dithionite or enzymatically using ferredoxin reductase and NADPH. For kinetic analysis, initial reaction rates should be measured across varying concentrations of both ferredoxin (0.1-50 μM) and NADP+ (1-500 μM) to determine Km, Vmax, and kcat values. Alternative approaches include a coupled assay system where NADPH produced by BCE_5065 drives a secondary reaction with a chromogenic or fluorogenic readout, providing enhanced sensitivity for low-activity preparations. Oxygen sensitivity should be addressed by conducting assays under anaerobic conditions or including an oxygen-scavenging system (glucose oxidase/catalase) in the reaction mixture. Comparison of the enzymatic efficiency of BCE_5065 with related reductases can be achieved by calculating the catalytic efficiency (kcat/Km) under standardized conditions, allowing for meaningful cross-enzyme analysis .

How does BCE_5065 interact with other proteins in the B. cereus redox network?

BCE_5065 participates in complex protein-protein interactions within the B. cereus redox network, serving as an integral component of electron transfer pathways. Research on related ferredoxin reductases in B. cereus has demonstrated that these enzymes exhibit highly specific interactions with their redox partners, suggesting that BCE_5065 likely recognizes distinct structural features on its ferredoxin substrates . The interaction interface between BCE_5065 and ferredoxin involves complementary electrostatic surfaces, with the predominantly positively charged surface on BCE_5065 interacting with the negatively charged surface of ferredoxin, positioning the prosthetic groups of both proteins at an optimal distance for efficient electron transfer. While TrxR-like FNRs in B. cereus have been shown to interact with flavodoxin-like proteins such as NrdI, BCE_5065 may exhibit a different partner specificity profile that defines its unique role in redox metabolism . Protein-protein docking studies, co-immunoprecipitation experiments, and surface plasmon resonance analyses could reveal the binding kinetics and specificity determinants of BCE_5065 interactions. Understanding these interaction networks is crucial for mapping the complete electron flow pathways in B. cereus and identifying potential metabolic bottlenecks or regulatory nodes. Comparative studies with other B. cereus reductases, such as the three characterized TrxR-like FNRs (FNR1-3), would provide valuable insights into the functional partitioning of electron transfer tasks among these related but distinct enzymes .

What structural modifications can enhance BCE_5065 catalytic efficiency for biotechnological applications?

Strategic structural modifications can significantly enhance the catalytic efficiency of BCE_5065 for specialized biotechnological applications. Site-directed mutagenesis targeting residues in the NADP+-binding domain might increase the affinity for this cofactor, potentially creating variants with lower Km values and improved catalytic efficiency under limited cofactor availability. Mutations at the ferredoxin-binding interface could broaden substrate specificity, allowing engineered BCE_5065 to accept electrons from non-native ferredoxins or even artificial electron donors, expanding its utility in synthetic biology applications. Research on related enzymes has shown that altering the orientation of the NADPH-binding domain relative to the FAD-binding domain can impact catalytic properties, suggesting that introduction of flexible linkers or constraining elements might optimize the conformational dynamics of BCE_5065 during catalysis . Temperature stability could be enhanced through the introduction of additional disulfide bonds or salt bridges in regions prone to unfolding, creating variants suitable for industrial processes operating at elevated temperatures. Comparative analysis with homologous reductases that exhibit distinctive kinetic properties, such as the differences observed between TrxR and TrxR-like FNRs in B. cereus, provides insights into which structural elements might be exchanged to confer desired catalytic behaviors . Protein engineering approaches such as directed evolution or semi-rational design combining computational predictions with high-throughput screening would be particularly effective for developing BCE_5065 variants with enhanced performance characteristics for specific biotechnological applications.

How does BCE_5065 compare to homologous enzymes in other bacterial species in terms of substrate specificity?

BCE_5065 from Bacillus cereus exhibits distinctive substrate specificity patterns that differentiate it from homologous enzymes in other bacterial species. Comparative analysis reveals that while BCE_5065 belongs to the ferredoxin--NADP reductase type 2 family, its substrate recognition profile likely differs from related enzymes in organisms like Escherichia coli or Pseudomonas species . In B. cereus, studies of related reductases have identified significant variations in catalytic efficiencies and turnover rates with different electron donor/acceptor pairs, suggesting that BCE_5065 has evolved specialized interaction surfaces that optimize electron transfer with specific ferredoxin partners native to B. cereus . The lack of the CXXC catalytic motif in BCE_5065, which is present in true thioredoxin reductases, structurally explains its inability to efficiently reduce thioredoxins, demonstrating how subtle structural differences drive major functional divergence among related oxidoreductases . Notably, within the Bacillus genus, species-specific variations in the electrostatic surface properties of ferredoxin reductases influence their ability to recognize ferredoxins from different sources, with BCE_5065 likely exhibiting highest activity with ferredoxins from closely related Bacillus species. The substrate specificity of BCE_5065 represents an evolutionary adaptation to the specific electron transfer requirements of B. cereus metabolism, potentially contributing to this organism's ability to thrive in its ecological niche. Detailed kinetic analysis with a panel of ferredoxins from diverse bacterial sources would provide quantitative insights into the substrate selectivity profile of BCE_5065 compared to its homologs.

How can researchers maintain the stability and activity of purified BCE_5065 during storage and experiments?

Maintaining the stability and activity of purified BCE_5065 requires careful attention to storage conditions and handling protocols. For long-term storage, BCE_5065 should be maintained at -80°C in a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 50% glycerol to prevent freeze-thaw damage and oxidative inactivation . When preparing working stocks, avoid repeated freeze-thaw cycles by aliquoting the enzyme preparation into single-use volumes before freezing. During experimental procedures, maintaining BCE_5065 on ice or at 4°C and minimizing exposure to light will help preserve the integrity of the FAD cofactor, which is sensitive to photodegradation. The inclusion of reducing agents such as 1 mM DTT or 2 mM β-mercaptoethanol in all working buffers protects cysteine residues from oxidation and helps maintain the native conformation of the enzyme. Protein concentration should be kept above 0.1 mg/mL to prevent surface-induced denaturation, with the addition of 0.1 mg/mL BSA as a stabilizing agent if more dilute solutions are required. Spectroscopic monitoring of the characteristic FAD absorption peaks at approximately 375 and 450 nm provides a convenient method to assess cofactor retention and protein integrity over time. For applications requiring extended experiment durations, progressive loss of activity can be mitigated by supplementing the reaction mixture with fresh FAD (1-10 μM) and including an ATP-regenerating system if NADPH is being utilized in coupled assays.

What are common challenges in recombinant BCE_5065 expression and how can they be resolved?

Researchers frequently encounter several challenges when expressing recombinant BCE_5065 that can be addressed through specific optimization strategies. Insufficient soluble protein expression often results from rapid accumulation as inclusion bodies, which can be mitigated by reducing the induction temperature to 16-20°C, decreasing IPTG concentration to 0.1-0.2 mM, and extending the induction period to 16-24 hours to favor proper folding . Inadequate FAD incorporation, indicated by weak yellow coloration of the purified protein, can be addressed by supplementing the culture medium with riboflavin (10 μM) and including FAD (5-10 μM) in the lysis buffer. Proteolytic degradation during expression or purification, evidenced by multiple bands on SDS-PAGE, can be minimized by using protease-deficient host strains, including protease inhibitor cocktails in all buffers, and maintaining samples at 4°C throughout processing. Low enzymatic activity despite successful expression may indicate improper folding, which can be improved by co-expressing molecular chaperones such as GroEL/GroES or DnaK/DnaJ/GrpE systems. For expression constructs yielding toxic effects on the host cells, employing tightly regulated expression systems like pET with T7 lysozyme co-expression or utilizing specialized expression strains with reduced basal expression can improve culture viability and final protein yields. Codon optimization of the BCE_5065 sequence for E. coli expression can resolve translation inefficiency caused by rare codon usage, particularly for arginine and leucine codons that differ between Bacillus and E. coli.

How can site-directed mutagenesis be effectively applied to study BCE_5065 structure-function relationships?

Site-directed mutagenesis represents a powerful approach for investigating the structure-function relationships of BCE_5065, providing insights into catalytic mechanisms and substrate specificity determinants. When designing a mutagenesis strategy, researchers should prioritize residues at the NADP+-binding site, FAD-binding pocket, and ferredoxin-interaction interface based on sequence alignments with characterized homologs and available structural information . The QuikChange mutagenesis protocol using complementary primers containing the desired mutation offers an efficient method for introducing specific amino acid substitutions, with high-fidelity polymerases like Pfu Ultra or Q5 minimizing unintended mutations. Conservative substitutions (e.g., Asp to Glu) provide information about the importance of specific chemical properties, while non-conservative changes (e.g., Asp to Ala) can reveal the essentiality of particular functional groups. Alanine-scanning mutagenesis of surface residues at the predicted ferredoxin-binding interface can systematically map the interaction determinants controlling partner specificity. When investigating the FAD-binding domain, mutations should be evaluated not only for effects on catalytic activity but also for impacts on FAD incorporation, which can be assessed spectrophotometrically by comparing the A280/A450 ratios of wild-type and mutant proteins. Based on studies of related reductases, investigating the role of interdomain dynamics through mutations at domain interfaces could provide insights into the conformational changes that occur during catalysis . For comprehensive analysis, mutant proteins should be characterized through steady-state kinetics with varying concentrations of both substrates (ferredoxin and NADP+), thermostability assays, and protein-protein interaction studies to fully understand how specific residues contribute to the diverse functional properties of BCE_5065.

How do Ferredoxin--NADP reductases from Bacillus cereus compare with those from other bacterial phyla?

Ferredoxin--NADP reductases from Bacillus cereus display distinctive features when compared with homologous enzymes from other bacterial phyla, reflecting evolutionary adaptations to different metabolic requirements and ecological niches. While BCE_5065 belongs to the ferredoxin--NADP reductase type 2 family that is widespread among Gram-positive bacteria, significant structural and functional variations exist between B. cereus FNRs and those from Gram-negative bacteria or cyanobacteria . Unlike plant-type FNRs found in cyanobacteria and plastids, which possess a distinct domain organization and catalytic mechanism, B. cereus FNRs belong to the glutathione reductase-like FNR subfamily, exhibiting structural homology to TrxR but lacking the CXXC catalytic motif . Studies of TrxR-like FNRs in B. cereus have revealed that they can function in diverse biochemical contexts, with FNR2 serving as an NrdI reductase in ribonucleotide reduction, FNR3 functioning as a bacillithiol disulfide reductase, and FNR1 homologs in related organisms acting as iron-uptake oxidoreductases . This functional diversity within a single organism contrasts with the more specialized roles typically observed for FNRs in Gram-negative bacteria. Sequence analysis and phylogenetic studies indicate that B. cereus FNRs form distinct clades within the broader family of bacterial oxidoreductases, suggesting lineage-specific evolutionary trajectories that have shaped their current functional properties. The comparative biochemistry of these enzymes provides valuable insights into how oxidoreductases have diversified to fulfill specialized roles while maintaining core catalytic capabilities.

What is known about the evolutionary origins of BCE_5065 and related reductases?

The evolutionary origins of BCE_5065 and related reductases reflect complex patterns of gene duplication, functional divergence, and potential horizontal gene transfer events throughout bacterial evolution. Phylogenetic analyses place BCE_5065 within the GR-like FNR subfamily, which is believed to have emerged through ancient duplication events from a common ancestor shared with thioredoxin reductases, followed by loss of the CXXC catalytic motif and acquisition of surface features favoring ferredoxin interaction over thioredoxin binding . The distribution of TrxR-like FNRs across bacterial phyla suggests they were present in the last common ancestor of Firmicutes, with subsequent lineage-specific gene duplication events leading to the diversity observed in modern Bacillus species, which typically encode multiple FNR paralogs with specialized functions . Sequence comparison studies reveal that BCE_5065 shares core structural features with homologs in other Firmicutes while exhibiting species-specific adaptations in substrate-binding regions that reflect the particular electron transfer requirements of B. cereus metabolism. Comparative genomic analyses have identified synteny patterns suggesting that BCE_5065 belongs to an ancient gene cluster involved in redox metabolism, with neighboring genes often encoding electron transfer proteins or metabolic enzymes requiring reducing power. Analysis of selection pressures acting on BCE_5065 and its homologs indicates ongoing adaptive evolution, particularly at residues involved in substrate recognition, suggesting continued refinement of function in response to changing metabolic demands. These evolutionary patterns underscore how redox enzymes like BCE_5065 have been shaped by both vertical inheritance and functional specialization to create the diverse array of ferredoxin reductases observed in contemporary bacteria.

How does the substrate specificity of BCE_5065 reflect its evolutionary adaptation to B. cereus metabolism?

The substrate specificity profile of BCE_5065 represents a fine-tuned evolutionary adaptation to the particular metabolic requirements of Bacillus cereus. Through progressive sequence divergence following gene duplication events, BCE_5065 has developed specific surface features that optimize interaction with the ferredoxins endemic to B. cereus, creating an electron transfer system tailored to this organism's metabolic needs . Research on related reductases has demonstrated that even closely related enzymes can exhibit distinct substrate preferences, as evidenced by the different activities of B. cereus FNRs toward various electron acceptors, suggesting that BCE_5065's specificity pattern reflects selection for a particular metabolic role . The alignment of BCE_5065's catalytic properties with the redox potential requirements of B. cereus metabolism represents a co-evolutionary process, where the enzyme's kinetic parameters have been optimized for the concentrations of substrates and products typically encountered in its cellular environment. Comparative analysis of BCE_5065 with homologs from closely related Bacillus species reveals how subtle sequence variations translate into functional differences that may contribute to species-specific metabolic capabilities, potentially influencing ecological adaptation. Unlike the TrxR system that operates primarily in response to oxidative stress through disulfide reduction, BCE_5065 likely participates in metabolic pathways involving iron-sulfur proteins, reflecting the importance of these pathways in B. cereus physiology . This functional partitioning between different reductase systems represents a sophisticated evolutionary solution to the challenge of maintaining distinct but interconnected electron transfer pathways, allowing for precise regulation of redox metabolism in response to changing environmental conditions.