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.

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.

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.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners.

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 µM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.

New azole derivatives of [17(20)E]-21-norpregnene: Synthesis and inhibition of prostate carcinoma cell growth.

A number of isoxazole, 1,2,3-triazole, tetrazole, and 1,2,4-oxadiazole derivatives of [17(20)E]-21-norpregnene comprising 3ß-hydroxy-5-ene and 3-oxo-4-ene fragments were prepared. Among the key steps for the synthesis of isoxazoles, 1,2,3-triazoles, and tetrazoles were (i) 1,3-dipolar cycloaddition of nitrile oxides or azides to acetylenes or nitriles and ii) dehydration of 17ß-hydroxy-17α-methylene-azoles to [17(20)E]-21-norpregnene derivatives. 1,2,4-Oxadiazoles were prepared through the formation of acetimidamides. Potency of the synthesized compounds to inhibit CYP17A1 and to suppress growth of prostate carcinoma cells was investigated. Among the new azole derivatives, four compounds were found possessing high anti-proliferative activity.

Different inhibitory effects of azole-containing drugs and pesticides on CYP2C9 polymorphic forms: An in vitro study.

CYP2C9 plays a major role in drug metabolism. It is highly polymorphic and among the variants, CYP2C9*2 and CYP2C9*3 have been known to encode the protein with moderately to markedly reduced catalytic activity. Azole antifungals are among the most frequently used drugs in human pharmacotherapy and represent a widely used class of pesticides to which humans are inevitably exposed. Due to the similarities in CYP organization throughout species, azoles can interact not only with the target fungal CYP51 substrate-binding site but can also modulate the catalytic activity of human cytochrome P450s, including CYP2C9, causing severe adverse effects. In the present study the potency of azole-containing drugs and pesticides to inhibit recombinant wild-type CYP2C9*1 and the allelic variants CYP2C9*2 and CYP2C9*3 was evaluated. Significant differences were found in their affinity to CYP2C9*1, CYP2C9*2, and CYP2C9*3 as well as in the catalytic activity of CYP2C9 allelic variants. Moreover, addition of cytochrome b5 resulted in a decrease of CYP2C9*3 activity to diclofenac in a concentration-dependent manner. Increasing the knowledge of how azoles influence polymorphic variants of CYP2C9 could help individualize drug treatment, leading to optimization of the selection of drugs and doses for individuals based on genetic information.