Skip to main content
SpringerLink
Log in

Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from Candida glabrata (Rec-CgHMGR) Obtained by Heterologous Expression, as a Novel Therapeutic Target Model for Testing Synthetic Drugs

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k m and V max for the HMG-CoA were 6.5 μM and 2.26 × 10−3 μM min−1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5 μM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Durr, I. F., & Rudney, H. (1960). The reduction of beta-hydroxy-beta-methyl-glutaryl coenzyme A to mevalonic acid. Journal of Biological Chemistry, 235, 2572–2578.

    CAS  Google Scholar 

  2. Burg, J. S., & Espenshade, P. J. (2011). Regulation of HMG-CoA reductase in mammals and yeast. Progress in Lipid Research, 50, 403–410.

    Article  CAS  Google Scholar 

  3. Pichandi, S., Pasupathi, P., Raoc, Y. Y., Farook, J., Ambika, A., Ponnusha, B. S., Subramaniyam, S., Virumandy, R., & Subramaniyam, B. (2011). The role of statin drugs in combating cardiovascular diseases. International Journal of Current Science, 1, 47–56.

    Google Scholar 

  4. Aronow, W. S. (2015). Treatment of hypercholesterolemia in 2015. American Journal of Therapeutics. doi:10.1097/MJT.0000000000000358.

    Google Scholar 

  5. Westermayer, C., & Macreadie, I. G. (2007). Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA in Candida glabrata. FEMS Yeast Research, 7, 436–441.

    Article  Google Scholar 

  6. Bochar, D. A., Stauffacher, C. V., & Rodwell, V. W. (1999). Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Molecular Genetics and Metabolism, 66, 122–127.

    Article  CAS  Google Scholar 

  7. Friesen, J. A., & Rodwell, V. W. (2004). The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases. Genome Biology, 5, 248–254.

    Article  Google Scholar 

  8. Istvan, E. S., & Deisenhofer, J. (2000). The structure of the catalytic portion of human HMG-CoA reductase. Biochimica et Biophysica Acta, 1529, 9–18.

    Article  CAS  Google Scholar 

  9. Istvan, E. S., Palnitkar, M., Buchanan, S. K., & Deisenhofer, J. (2000). Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis. EMBO Journal, 19, 819–830.

    Article  CAS  Google Scholar 

  10. Nash, J. D., Burgess, D. S., & Talbert, R. L. (2002). Effect of fluvastatin and pravastatin, HMG-CoA reductase inhibitors, on fluconazole activity against Candida albicans. Journal of Medical Microbiology, 51, 105–109.

    Article  CAS  Google Scholar 

  11. Argüelles, N., Sánchez-Sandoval, E., Mendieta, A., Villa-Tanaca, L., Garduño-Siciliano, L., Jiménez, F., Cruz, M. D. C., Medina-Franco, J. L., Chamorro-Cevallos, G., & Tamariz, J. (2010). Design, synthesis, and docking of highly hypolipidemic agents: Schizosaccharomyces pombe as a new model for evaluating α-asarone-based HMG-CoA reductase inhibitors. Bioorganic & Medicinal Chemistry, 18, 4238–4248.

    Article  Google Scholar 

  12. Andrade-Pavón, D., Sánchez-Sandoval, E., Rosales-Acosta, B., Ibarra, J. A., Tamariz, J., Hernández-Rodríguez, C., & Villa-Tanaca, L. (2014). The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: a proposal as a therapeutic target and as a study model. Revista Iberoamericana de Micología, 31, 81–85.

    Article  Google Scholar 

  13. Pfaller, M. A., & Diekema, D. J. (2007). Epidemiology of invasive candidiasis: a persistent public health problem. Clinical Microbiology Reviews, 20, 133–163.

    Article  CAS  Google Scholar 

  14. Hitchcock, C. A., Pye, G. W., Troke, P. F., Johnson, E. M., & Warnock, D. W. (1993). Fluconazole resistance in Candida glabrata. Antimicrobial Agents and Chemotherapy, 37, 1962–1965.

    Article  CAS  Google Scholar 

  15. Hernáez, M. L., Pla, J., & Nombela, C. (1997). Molecular and genetic aspects azole resistance in Candida albicans. Revista Iberoamericana de Micología, 14, 150–154.

    Google Scholar 

  16. Pasqualotto, A. C., & Denning, D. W. (2008). New and emerging treatments for fungal infections. Journal of Antimicrobial Chemotherapy, 61, 19–30.

    Article  Google Scholar 

  17. Dujon, B., Sherman, D., Fischer, G., Durrens, P., Casaregola, S., Lafontaine, I., De Montigny, J., Marck, C., Neuvéglise, C., Talla, E., Goffard, N., Frangeul, L., Aigle, M., Anthouard, V., Babour, A., Barbe, V., Barnay, S., Blanchin, S., Beckerich, J. M., Beyne, E., Bleykasten, C., Boisramé, A., Boyer, J., Cattolico, L., Confanioleri, F., De Daruvar, A., Despons, L., Fabre, E., Fairhead, C., Ferry-Dumazet, H., Groppi, A., Hantraye, F., Hennequin, C., Jauniaux, N., Joyet, P., Kachouri, R., Kerrest, A., Koszul, R., Lemaire, M., Lesur, I., Ma, L., Muller, H., Nicaud, J. M., Nikolski, M., Oztas, S., Ozier-Kalogeropoulos, O., Pellenz, S., Potier, S., Richard, G. F., Straub, M. L., Suleau, A., Swennen, D., Tekaia, F., Wésolowski-Louvel, M., Westhof, E., Wirth, B., Zeniou-Meyer, M., Zivanovic, I., Bolotin-Fukuhara, M., Thierry, A., Bouchier, C., Caudron, B., Scarpelli, C., Gaillardin, C., Weissenbach, J., Wincker, P., & Souciet, J. L. (2004). Genome evolution in yeasts. Nature, 430, 35–44.

    Article  Google Scholar 

  18. Durfee, T., Nelson, R., Baldwin, S., Plunkett 3rd, G., Burland, V., Mau, B., Petrosino, J. F., Qin, X., Muzny, D. M., Ayele, M., Gibbs, R. A., Csörgo, B., Pósfai, G., Weinstock, G. M., & Blattner, F. R. (2008). The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. Journal of Bacteriology, 19, 2597–2606.

    Article  Google Scholar 

  19. Grodberg, J., & Dunn, J. J. (1988). OmpT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. Journal of Bacteriology, 170, 1245–1253.

    Article  CAS  Google Scholar 

  20. Smith, T. F., & Waterman, M. S. (1981). Identification of common molecular subsequences. Journal of Molecular Biology, 147, 195–197.

    Article  CAS  Google Scholar 

  21. Sigrist, C. J., de Castro, E., Cerutti, L., Cuche, B. A., Hulo, N., Bridge, A., Bougueleret, L., & Xenarios, I. (2013). New and continuing developments at PROSITE. Nucleic Acids Research, 41, 344–347.

    Article  Google Scholar 

  22. Hirokawa, T., Boon-Chieng, S., & Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane protein. Bioinformatics, 14, 378–379.

    Article  CAS  Google Scholar 

  23. Nakai, K., & Horton, P. (1999). PSORT: a program for detecting the sorting signals of proteins and predicting their subcellular localization. Trends in Biochemical Sciences, 24, 34–35.

    Article  CAS  Google Scholar 

  24. Webb, B., & Sali, A. (2014). Comparative protein structure modeling using Modeller. Current Protocols in Bioinformatics, 5(6), 1–5.6.32.

    Google Scholar 

  25. Osipovitch, M., Lambrecht, M., Baker, C., Madha, S., Mills, J. L., Craig, P. A., & Bernstein, H. J. (2015). Automated protein motif generation in the structure-based protein function prediction tool ProMOL. Journal of Structural and Functional Genomics, 16, 101–111.

    Article  CAS  Google Scholar 

  26. Green, M. R., & Sambrook, J. (2012). A molecular cloning: a laboratory manual (4th ed.). NJ: Cold Spring Harbor.

    Google Scholar 

  27. Ibarra, J. A., García-Zacarías, C. M., Lara-Ochoa, C., Carabarin-Lima, A., Tecpanecatl-Xihuitl, J. S., Pérez-Rueda, E., Martínez-Laguna, Y., & Puente, J. L. (2013). Further characterization of functional domains of PerA, role of amino and carboxy terminal domains in DNA binding. PloS One, 8, e56977.

    Article  CAS  Google Scholar 

  28. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  29. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 351–355.

    Article  Google Scholar 

  30. Bischoff, K. M., & Rodwell, V. W. (1995). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli. Journal of Bacteriology, 178, 19–23.

    Article  Google Scholar 

  31. Kleinsek, D. A., Dugan, R. E., Baker, T. A., & Porter, J. W. (1981). 3-hydroxy-3-methylglutaryl-CoA reductase from rat liver. Methods in Enzymology, 71, 462–479.

    Article  CAS  Google Scholar 

  32. Bates, R. G. (1964). Determination of pH: theory and practice. NY: Wiley.

    Google Scholar 

  33. Whaley, S. G., & Rogers, P. D. (2016). Azole resistance in Candida glabrata. Current Infectious Disease Reports, 18, 41.

    Article  Google Scholar 

  34. Campoy, S., & Adrio, J. L. (2016). Antifungal. Biochemical Pharmacology. doi:10.1016/j.bcp.2016.11.019.

    Google Scholar 

  35. Mayer, R. J., Debouck, C., & Metcalf, B. W. (1998). Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli. Archives and Biochemistry and Biophysics, 267, 110–118.

    Article  Google Scholar 

  36. Juárez-Montiel, M., Ibarra, J. A., Chávez-Camarillo, G., Hernández-Rodríguez, C., & Villa-Tanaca, L. (2014). Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis. Applied Biochemistry and Biotechnology, 172, 2530–2539.

    Article  Google Scholar 

  37. Rao, D. V., Ramu, C. T., Rao, J. V., Narasu, M. L., & Rao, A. K. (2009). Cloning, high expression and purification of recombinant human interferon-beta-1b in Escherichia coli. Applied Biochemistry and Biotechnology, 158, 140–154.

    Article  Google Scholar 

  38. Wang, Y., Qian, S., Meng, G., & Zhang, S. (2001). Cloning and expression of L-asparaginase gene in Escherichia coli. Applied Biochemistry and Biotechnology, 95, 93–101.

    Article  CAS  Google Scholar 

  39. Brondyk, W. H. (2009). Selecting an appropriate method for expressing a recombinant protein. Methods in Enzymology, 463, 131–147.

    Article  CAS  Google Scholar 

  40. Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27, 297–306.

    Article  CAS  Google Scholar 

  41. Croxen, R., Goosey, M. W., Keon, J. P., & Hargreaves, J. A. (1994). Isolation of an Ustilago maydis gene encoding 3-hydroxy-3-methylglutaryl-coenzyme reductase and expression of C-terminal-truncated forms in Escherichia coli. Microbiology, 140, 2363–2370.

    Article  CAS  Google Scholar 

  42. Theivagt, A. E., Amanti, E. N., Beresford, N. J., Tabernero, L., & Friesen, J. A. (2006). Characterization of an HMG-CoA reductase from Listeria monocytogenes that exhibits dual coenzyme specificity. Biochemistry, 45, 14397–14406.

    Article  CAS  Google Scholar 

  43. Takahashi, S., Kuzuyama, T., & Seto, H. (1999). Purification, characterization and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids. Journal of Bacteriology, 181, 256–263.

    Google Scholar 

  44. Hurtado-Guerrero, R., Peña-Díaz, J., Montalvetti, A., Ruiz-Pérez, L. M., & González-Pacanowska, D. (2002). Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase. FEBS Letters, 510, 141–144.

    Article  Google Scholar 

  45. Istvan, E. S., & Deisenhofer, J. (2001). Structural mechanism for statin inhibition of HMG-CoA reductase. Science, 292, 1160–1164.

    Article  CAS  Google Scholar 

  46. Pedersen, T. R., & Tobert, J. A. (2004). Simvastatin: a review. Expert of Opinion on Pharmacotherapy, 5, 2583–2596.

    Article  CAS  Google Scholar 

  47. Mendieta, A., Jiménez, F., Garduño-Siciliano, L., Mojica-Villegas, A., Rosales-Acosta, B., Villa-Tanaca, L., Chamorro-Cevallos, G., Medina-Franco, J. L., Meurice, N., Gutiérrez, R. U., Montiel, L. E., Cruz Mdel, C., & Tamariz, J. (2014). Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors. Bioorganic & Medicinal Chemistry, 22, 5871–5882.

    Article  CAS  Google Scholar 

  48. Chin, N. X., Weitzman, I., & Della-Latta, P. (1997). In vitro activity of fluvastatin, a cholesterol-lowering agent, and synergy with fluconazole and itraconazole against Candida species and Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy, 41, 850–852.

    CAS  Google Scholar 

  49. Nyilasi, I., Kocsubé, S., Pesti, M., Lukács, G., Papp, T., & Vágvölgyi, C. (2010a). In vitro interactions between primycin and different statins in their effects against some clinically important fungi. Journal of Medical Microbiology, 59, 200–205.

    Article  CAS  Google Scholar 

  50. Nyilasi, I., Kocsubé, S., & Krizsán, K. (2010b). In vitro synergistic interactions of the effects of various statins and azoles against some clinically important fungi. FEMS Microbiology Letters, 307, 175–184.

    Article  CAS  Google Scholar 

  51. Singh, S., Dinesh, N., Kaur, P. K., & Shamiulla, B. (2014). Ketanserin, an antidepressant, exerts its antileishmanial action via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme of Leishmania donovani. Parasitology Research, 113, 2161–2168.

    Article  Google Scholar 

  52. Chen, J. B., Chern, T. R., Wei, T. T., Chen, C. C., Lin, J. H., & Fang, J. M. (2013). Design and synthesis of dual-action inhibitors targeting histone deacetylases and 3-hydroxy-3-methylglutaryl coenzyme A reductase for cancer treatment. Journal of Medical Chemistry, 56, 3645–3655.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Yuridia Mercado for helpful technical advice. Dr. Ravi Pathak is very much appreciated for proofreading this manuscript.

Author information

Authors and Affiliations

  1. Laboratorio de Genética Microbiana, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prol. de Carpio y Plan de Ayala. Col. Sto. Tomás, 11340, Ciudad de México, DF, Mexico

    Dulce Andrade-Pavón, César Hernández-Rodríguez, J. Antonio Ibarra & Lourdes Villa-Tanaca

  2. Laboratorio de Investigación en Bioquímica, Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, Casco de Santo Tomás, 11340, Ciudad de México, Mexico

    Roberto I. Cuevas-Hernández & José G. Trujillo-Ferrara

Authors
  1. Dulce Andrade-Pavón
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roberto I. Cuevas-Hernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José G. Trujillo-Ferrara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. César Hernández-Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Antonio Ibarra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lourdes Villa-Tanaca
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to J. Antonio Ibarra or Lourdes Villa-Tanaca.

Ethics declarations

Funding

This work was supported by CONACYT [133695] and SIP-IPN [20161403, 20161245; 20150981 and 20150612]. DMA-P and RIC-H were recipients of fellowships from CONACyT and BEIFI-IPN. JAI, CHR, and LVT received support from COFAA-IPN, EDI-IPN, and SNI CONACyT. JAI and LVT were hired through “Programa Institucional de Contratación de Personal Académico de Excelencia (PICPAE) IPN.”

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(DOCX 367 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Andrade-Pavón, D., Cuevas-Hernández, R.I., Trujillo-Ferrara, J.G. et al. Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from Candida glabrata (Rec-CgHMGR) Obtained by Heterologous Expression, as a Novel Therapeutic Target Model for Testing Synthetic Drugs. Appl Biochem Biotechnol 182, 1478–1490 (2017). https://doi.org/10.1007/s12010-017-2412-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2412-9

Keywords

Search

Navigation