Ligand-binding characteristics of CYP51 Mycobacterium tuberculosis in relation to marine steroid compounds

CYP51 steroid-14α-demethylases are members of a large superfamily of cytochrome P450 enzymes found in all kingdoms of living organisms, and catalyze the 14α-demethylation reaction of a number of natural steroids, including lanosterol, obtusifoliol, and 24,25-dihydrolanosterol. CYP51 are important components of the eukaryotic steroid biosynthetic chain, and thus represent one of the main targets for antifungal therapy. A 14α-demethylase CYP51 homologous gene has also been found in the genome of Mycobacterium tuberculosis. At the same time, M. tuberculosis lacks the de novo pathway for steroid biosynthesis. Conservation of CYP51 among the Mycobacterium genus and colocalization in the genome with 3Fe-4S ferredoxin Rv0763c, which maintains its catalytic activity in vitro, may indirectly indicate the involvement of MTCYP51 in a biochemical process important for mycobacteria. In order to characterize the specificity of the MTCYP51 active site to various compounds of isoprenoid nature, we obtained a highly purified MTCYP51 and, using spectrophotometric titration and surface plasmon resonance methods, studied the interaction of MTCYP51 with steroids from marine organisms obtained in the Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences. The investigated compounds represent a wide range of evolutionarily ancient isoprenoids. The results showed that MTCYP51 is able to bind structurally diverse steroid derivatives in the active site. The conducted studies suggest the biological role of MTCYP51 for pathogenic mycobacteria, which consists in the binding and possible metabolism of exogenous bioregulatory isoprenoids in vivo. 

1. World Health organization. Global tuberculosis report 2022. Geneva, 2022.

2. Cole, S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D. E., Gordon S. V. [et al.]. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998, vol. 393, no. 6685, pp. 537–544. https:// doi.org/10.1038/31159

3. Aoyama Y., Horiuchi T., Gotoh O., Noshiro M., Yoshida Y. CYP51-like gene of Mycobacterium tuberculosis actually encodes a P450 similar to eukaryotic CYP51. Journal of Biochemistry, 1998, vol. 124, no. 4, pp. 694–696. https://doi.org/10.1093/oxfordjournals.jbchem.a022167

4. Bellamine A., Mangla A. T., Nes W. D., Waterman M. R. Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences USA, 1999, vol. 96, no. 16, pp. 8937–8942. https://doi.org/10.1073/pnas.96.16.8937

5. Strushkevich, N., Usanov S. A., Park H.-W. Structural basis of human CYP51 inhibition by antifungal azoles. Journal of Molecular Biology, 2010, vol. 397, no. 4, pp. 1067–1078. https://doi.org/10.1016/j.jmb.2010.01.075

6. Lamb D. C., Lei L., Warrilow A. G. S., Lepesheva G. I., Mullins J. G. L., Waterman M. R., Kelly S. L. The first virally encoded cytochrome p450. Journal of Virology, 2009, vol. 83, no. 16, pp. 8266–8269. https://doi.org/10.1128/jvi.00289-09

7. DeJesus M. A., Gerrick E. R., Xu W., Park S. W., Long J. E., Boutte C. C., Rubin E. J. [et al.]. Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis. mBio, 2017, vol. 8, no. 1, pp. 1–17. https://doi.org/10.1128/mBio.02133-16

8. Wynn E. A., Dide-Agossou C., Reichlen M., Rossmassler K., Mubarak R. A., Reid J. J., Tabor S. T. [et al.]. Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice. bioRxiv [Preprint], 2023, March 08. Available at: https://www.biorxiv.org/content/10.1101/2023.03.06.531356v2. https://doi.org/10.1101/2023.03.06.531356

9. Gilep A., Varaksa T., Bukhdruker S., Kavaleuski A., Ryzhykau Y., Smolskaya S., Sushko T. Structural insights into 3Fe–4S ferredoxins diversity in M. tuberculosis highlighted by a first redox complex with P450. Frontiers in Molecular Biosciences, 2023, vol. 9, pp. 1–15. https://doi.org/10.3389/fmolb.2022.1100032

10. Eddine A. N., Kries J. P., Podust M. V., Warrier T., Kaufmann S. H. E., Podust L. M. X-ray structure of 4, 4′-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14α-demethylase (CYP51). Journal of Biological Chemistry, 2008, vol. 283, no. 22, pp. 15152–15159. https://doi.org/ 10.1074/jbc.M801145200

11. Podust L. M., Yermalitskaya L. V., Lepesheva G. I., Podust V. N., Dalmasso E. A., Waterman M. R. Estriol bound and ligand-free structures of sterol 14alpha-demethylase. Structure, 2004, vol. 12, no. 11, pp. 1937–1945. https://doi.org/10.1016/j. str.2004.08.009

12. Kaluzhskiy L., Ershov P., Yablokov E., Shkel T., Grabovec I., Mezentsev Y., Gnedenko O. [et al.]. Human lanosterol 14-alpha demethylase (CYP51A1) Is a putative target for natural flavonoid luteolin 7, 3′-disulfate. Molecules, 2021, vol. 26, no. 8, pp. 2237. https://doi.org/10.3390/molecules26082237

13. Lepesheva G. I., Nes W. D., Zhou W., Hill G. C., Waterman M. R. CYP51 from Trypanosoma brucei is obtusifoliolspecific. Biochemistry, 2004, vol. 43, no. 33, pp. 10789–10799. https://doi.org/10.1021/bi048967t

14. Schenkman J. B., Jansson I. Spectral analyses of cytochromes P450. Methods in molecular biology, 2006, vol. 320, pp. 11–18. https://doi.org/10.1385/1-59259-998-2:11

15. McLean K. J., Lafite P., Levy C., Cheesman M. R., Mast N., Pikuleva I. A., Leys D., Munro A. W. The structure of Mycobacterium tuberculosis CYP125: molecular basis for cholesterol binding in a P450 needed for host infection. Journal of Biological Chemistry, 2009, vol. 284, no. 51, pp. 35524–35533. https://doi.org/10.1074/jbc.M109.032706

16. Makarieva T. N., Stonik V. A., Kapustina I. I., Boguslavsky V. M., Dmitrenoik A. S., Kalinin V. I., Cordeiro M. L., Djerassi C. Biosynthetic studies of marine lipids. 42. Biosynthesis of steroid and triterpenoid metabolites in the sea cucumber Eupentacta fraudatrix. Steroids, 1993, vol. 58, no. 11, pp. 508–517. https://doi.org/10.1016/0039-128x(93)90026-j

17. Kicha A. A., Kalinovsky A. I., Malyarenko T. V., Ivanchina N. V., Dmitrenok P. S., Menchinskaya E. S., Yurchenko E. A. [et al.]. Cyclic steroid glycosides from the starfish Echinaster luzonicus: Structures and immunomodulatory activities. Journal of Natural Products, 2015, vol. 78, no. 6, pp. 1397–1405. https://doi.org/10.1021/acs.jnatprod.5b00332

18. Kicha A. A., Ha D. T., Ivanchina N. V., Malyarenko T. V., Kalinovsky A. I., Dmitrenok P. S., Ermakova S. P. Six new polyhydroxysteroidal glycosides, anthenosides S1–S6, from the starfish Anthenea sibogae. Chemistry & Biodiversity, 2018, vol. 15, no. 3, pp. 1–12. https://doi.org/10.1002/cbdv.201700553

19. Kicha A. A., Ha D. T., Malyarenko T. V., Kalinovsky A. I., Popov R. S., Malyarenko O. S., Thuy Tran T. T. Unusual polyhydroxylated steroids from the starfish Anthenoides laevigatus, collected of the coastal waters of Vietnam. Molecules, 2020, vol. 25, no. 6, pp. 1–12. https://doi.org/10.3390/molecules25061440

20. Ivanchina N. V., Kicha A. A., Malyarenko T. V., Ermolaeva S. D., Yurchenko E. A., E. A. Pislyagin, Minh C. V., Dmitrenok P. S. Granulatosides D, E and other polar steroid compounds from the starfish Choriaster granulatus. Their immunomodulatory activity and cytotoxicity. Natural Product Research, 2019, vol. 33, no. 18, pp. 2623–2630. https://doi.org/ 10.1080/14786419.2018.1463223

21. Ivanchina N. V., Kicha A. A., Huong T. T. T., Kalinovsky A. I., Dmitrenok P. S., Agafonova I. G., Long P. Q., Stonik V. A. Steroids, 2010, vol. 75, no. 12, pp. 897–904. https://doi.org/10.1016/j.steroids.2010.05.012

22. Tabakmakher K. M., Makarieva T. N., Denisenko V. A., Popov R. S., Dmitrenok P. S., Dyshlovoy S. A., Grebnev B. B. New trisulfated steroids from the Vietnamese marine sponge Halichondria vansoesti and their PSA expression and glucose uptake inhibitory activities. Marine Drugs, 2019, vol. 17, no. 8, pp. 455. https://doi.org/10.3390/md17080445

23. Tanious F. A., Nguyen B., Wilson W. D. Biosensor‐surface plasmon resonance methods for quantitative analysis of biomolecular interactions. Methods in Cell Biology, 2008, vol. 84, pp. 53–77. https://doi.org/10.1016/S0091-679X(07)84003-9

24. Lipschultz C. A., Li Y., Smith-Gill S. Experimental design for analysis of complex kinetics using surface plasmon resonance. Methods, 2000, vol. 20, no. 3, pp. 310–318. https://doi.org/10.1006/meth.1999.0924

25. Schenkman J. B., Sligar S. G., Cinti D. L. Substrate interaction with cytochrome P-450. Pharmacology & Therapeutics, 1981, vol. 12, no. 1, pp. 43–71. https://doi.org/10.1016/0163-7258(81)90075-9

26. Podust L. M., Poulos T. L., Waterman M. R. Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 2001, vol. 98, no. 6, pp. 3068–3073. https://doi.org/10.1073/pnas.061562898

27. Varaksa T., Bukhdruker S., Grabovec I., Marin E., Kavaleuski A., Gusach A., Kovalev K. [et al.]. Metabolic fate of human immunoactive sterols in Mycobacterium tuberculosis. Journal of Molecular Biology, 2021, vol. 433, no. 4, pp. 1–16. https://doi.org/ 10.1016/j.jmb.2020.166763

28. Vasilevskaya A. V., Yantsevich A. V., Sergeev G. V., Lemish A. P., Usanov S. A., Gilep A. A. Identification of Mycobacterium tuberculosis enzyme involved in vitamin D and 7-dehydrocholesterol metabolism. Journal of Steroid Biochemistry and Molecular Biology, 2017, vol. 169, pp. 202–209. https://doi.org/10.1016/j.jsbmb.2016.05.02