Biology subjects

Sushko, T.

Publications and source records attributed to Sushko, T..

4 recordsLinked to original sources

bioRxiv2025

True-atomic-resolution insights into the interactions of antibiotic rifampicin and rifamycin derivatives with orphan CYP143 of M.tuberculosis.

Analysis of drug-resistant strains of M.tuberculosis revealed a mutation in cytochrome P450 CYP143 gene located in the ESX-5 secretion cluster. The function of CYP143 is unknown. Available synteny information is insufficient to straightforwardly infer potential CYP143 substrates, because a full set of effector proteins/substrates exported by the ESX-5 secretion system has not yet been identified. Here we found that CYP143 is highly conserved among mycobacteria. We leverage the possible association of the G334A mutation in CYP143 with the development of resistance to test the frontline antitubercular drug rifampicin using purified proteins. Binding of rifampicin does not induce typical P450 spectral response, while the crystal structure at atomic resolution reveals the binding mode in the active site. The drug binds above the heme and adopts "closed" conformation of the ansa chain running almost parallel to the naphthoquinone core. In this conformation the oxygen atom bridging C12 and C29 of the macrocycle makes a hydrogen bond with a water molecule coordinating heme iron. The same binding mode was observed in crystal structures in complex with rifaximin, rifamycin S and two synthetic derivatives. In the ligand-bound state the protein retains an open conformation even in the presence of the redox partner, as evident from the crystal structure of the ternary complex. Despite binding close to the heme, no conversion was observed showing that either the rifamycins or the reduction system, or both, are insufficient to support a full catalytic cycle. These results pave the way to understand the development of rifampicin resistance/tolerance and the role of orphan CYPome of M.tuberculosis.

biochemistry↗

bioRxiv2022

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.

biochemistry↗

bioRxiv2020

Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s - Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ~1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

molecular biology↗

bioRxiv2020

A new twist of rubredoxin function in M. tuberculosis

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are ubiquitous iron-containing electron carriers that play important roles in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxin expression. However, their functions during M. tuberculosis infection is unknown. In the present work, we show that rubredoxin B (RubB) supports catalytic activity of mycobacterial 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 neutralized single charge on the surface of RubB did not dramatically decrease activity of studied CYPs, and isothermal calorimetry (ITC) experiments indicated that interactions are transient and not highly specific. Our findings suggest that a switch from ferredoxins to rubredoxins support CYP activity in M. tuberculosis-infected macrophages. Our electrochemical experiments suggest potential applications of RubB in biotechnology.

biochemistry↗