The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein-protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development.

Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. KEY POINTS: • Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display. • Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library. • A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases.

The current article aims to summarize all possible spectrum of protein-protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H2 ) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein-protein and multiprotein complexes are of great importance in enzyme-regulating and signal-transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain-specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue-dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero-complex formation with different protein partners, including cytochrome P450s are discussed.

A new twist of rubredoxin function in M. tuberculosis.

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are a ubiquitous [1Fe-0S] class of electron carriers that play an important role in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxins expression. However, their functions during M. tuberculosis infection are unknown. In the present work, we show that rubredoxin B (RubB) is able to efficiently shuttle electrons from cognate reductases, FprA and FdR to support catalytic activity of cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that not only neutralize single charge but also change the specific residues on the surface of RubB did not dramatically decrease activity of studied CYPs. Together with isothermal calorimetry (ITC) experiments, the obtained results suggest that interactions are transient and not highly specific. The redox potential of RubB is -264 mV vs. Ag/AgCl and the measured extinction coefficients are 9931 M-1cm-1 and 8371 M-1cm-1 at 380 nm and 490 nm, respectively. Characteristic parameters of RubB along with the discovered function might be useful for biotechnological applications. Our findings suggest that a switch from ferredoxins to rubredoxins might be crucial for M. tuberculosis to support CYPs activity during the infection.

Human Lanosterol 14-Alpha Demethylase (CYP51A1) Is a Putative Target for Natural Flavonoid Luteolin 7,3'-Disulfate.

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3'-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The Kd values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin-luteolin 7,3'-disulfate-showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3'-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.

Hydroxylation of Antitubercular Drug Candidate, SQ109, by Mycobacterial Cytochrome P450.

Spreading of the multidrug-resistant (MDR) strains of the one of the most harmful pathogen Mycobacterium tuberculosis (Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109 in vitro. A 1.25 Å-resolution crystal structure of the CYP124-SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the ω-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation.

Isatin (indole-2, 3-dione) is a non-peptide endogenous bioregulator exhibiting a wide spectrum of biological activity, realized in the cell via interactions with numerous isatin-binding proteins, their complexes, and (sub) interactomes. There is increasing evidence that isatin may be involved in the regulation of complex formations by modulating the affinity of the interacting protein partners. Recently, using Surface Plasmon Resonance (SPR) analysis, we have found that isatin in a concentration dependent manner increased interaction between two human mitochondrial proteins, ferrochelatase (FECH), and adrenodoxine reductase (ADR). In this study, we have investigated the affinity-enhancing effect of isatin on the FECH/ADR interaction. The SPR analysis has shown that FECH forms not only homodimers, but also FECH/ADR heterodimers. The affinity-enhancing effect of isatin on the FECH/ADR interaction was highly specific and was not reproduced by structural analogues of isatin. Bioinformatic analysis performed using three dimensional (3D) models of the interacting proteins and in silico molecular docking revealed the most probable mechanism involving FECH/isatin/ADR ternary complex formation. In this complex, isatin is targeted to the interface of interacting FECH and ADR monomers, forming hydrogen bonds with both FECH and ADR. This is a new regulatory mechanism by which isatin can modulate protein-protein interactions (PPI).

Thermodynamics of interactions between mammalian cytochromes P450 and b5.

Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent.

Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli.

Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system - cytochrome b5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm.

Computer-aided design of aptamers for cytochrome p450.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.

Structural characterization of human cholesterol 7α-hydroxylase.

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min-1, and the Michaelis constant (KM) was 78 ± 9 µM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

The effect of membrane composition on the interaction between human CYP51 and its flavonoid inhibitor - luteolin 7,3'-disulfate.

Cytochromes P450 (CYP) are a family of membrane proteins involved in the production of endogenous molecules and the metabolism of xenobiotics. It is well-known that the composition of the membrane can influence the activity and orientation of CYP proteins. However, little is known about how membrane composition affects the ligand binding properties of CYP. In this study, we utilized surface plasmon resonance and fluorescence lifetime analysis to examine the impact of membrane micro-environment composition on the interaction between human microsomal CYP51 (CYP51A1) and its inhibitor, luteolin 7,3'-disulphate (LDS). We observed that membranes containing cholesterol or sphingomyelin exhibited the lowest apparent equilibrium dissociation constant for the CYP51A1-LDS complex. Additionally, the tendency for relation between kinetic parameters of the CYP51A1-LDS complex and membrane viscosity and overall charge was observed. These findings suggest that the specific composition of the membrane, particularly the presence of cholesterol and sphingomyelin, plays a vital role in regulating the interaction between CYP enzymes and their ligands.

Improving the Efficiency of Electrocatalysis of Cytochrome P450 3A4 by Modifying the Electrode with Membrane Protein Streptolysin O for Studying the Metabolic Transformations of Drugs.

In the present work, screen-printed electrodes (SPE) modified with a synthetic surfactant, didodecyldimethylammonium bromide (DDAB) and streptolysin O (SLO) were prepared for cytochrome P450 3A4 (CYP3A4) immobilization, direct non-catalytic and catalytic electrochemistry. The immobilized CYP3A4 demonstrated a pair of redox peaks with a formal potential of -0.325 ± 0.024 V (vs. the Ag/AgCl reference electrode). The electron transfer process showed a surface-controlled mechanism ("protein film voltammetry") with an electron transfer rate constant (ks) of 0.203 ± 0.038 s-1. Electrochemical CYP3A4-mediated reaction of N-demethylation of erythromycin was explored with the following parameters: an applied potential of -0.5 V and a duration time of 20 min. The system with DDAB/SLO as the electrode modifier showed conversion of erythromycin with an efficiency higher than the electrode modified with DDAB only. Confining CYP3A4 inside the protein frame of SLO accelerated the enzymatic reaction. The increases in product formation in the reaction of the electrochemical N-demethylation of erythromycin for SPE/DDAB/CYP3A4 and SPE/DDAB/SLO/CYP3A4 were equal to 100 ± 22% and 297 ± 7%, respectively. As revealed by AFM images, the SPE/DDAB/SLO possessed a more developed surface with protein cavities in comparison with SPE/DDAB for the effective immobilization of the CYP3A4 enzyme.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4.

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein-Ligand Interactions of CYP51 of Candida krusei Azole-Resistant Fungal Strain.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.

Structural insights into the effects of glycerol on ligand binding to cytochrome P450.

New antitubercular drugs are vital due to the spread of resistant strains. Carbethoxyhexyl imidazole (CHImi) inhibits cytochrome P450 CYP124, which is a steroid-metabolizing enzyme that is important for the survival of Mycobacterium tuberculosis in macrophages. The available crystal structure of the CYP124-CHImi complex reveals two glycerol molecules in the active site. A 1.15 Šresolution crystal structure of the glycerol-free CYP124-CHimi complex reported here shows multiple conformations of CHImi and the CYP124 active site which were previously restricted by glycerol. Complementary molecular dynamics simulations show coherence of the ligand and enzyme conformations. Spectrophotometric titration confirmed the influence of glycerol on CHImi binding: the affinity decreases more than tenfold in glycerol-containing buffer. In addition, it also showed that glycerol has a similar effect on other azole and triazole CYP124 ligands. Together, these data show that glycerol may compromise structural-functional studies and impede rational drug-design campaigns.

At the crossroads of steroid hormone biosynthesis: the role, substrate specificity and evolutionary development of CYP17.

Cytochrome P450s play critical roles in the metabolism of various bioactive compounds. One of the crucial functions of cytochrome P450s in Chordata is in the biosynthesis of steroid hormones. Steroid 17alpha-hydroxylase/17,20-lyase (CYP17) is localized in endoplasmic reticulum membranes of steroidogenic cells. CYP17 catalyzes the 17alpha-hydroxylation reaction of delta4-C21 steroids (progesterone derivatives) and delta5-C21 steroids (pregnenolone derivatives) as well as the 17,20-lyase reaction producing C19-steroids, a key branch point in steroid hormone biosynthesis. Depending on CYP17 activity, the steroid hormone biosynthesis pathway is directed to either the formation of mineralocorticoids and glucocorticoids or sex hormones. In the present review, the current information on CYP17 is analyzed and discussed.

Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis.

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E0Ꞌ) of mutants did not significantly differ from the wild type protein and vary in the range of -264 to -231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.

Structural insights into 3Fe-4S ferredoxins diversity in M. tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis-Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high-affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.