Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties

Abstract

The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.

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

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Abbreviations

HSL:

mammalian hormone-sensitive lipase

PEG:

polyethylene glycol

SOE-PCR:

splicing by overlapping extension PCR

wtPMGL2:

wild-type PMGL2 esterase

REFERENCES

  1. 1

    Casas-Godoy, L., Duquesne, S., Bordes, F., Sandoval, G., and Marty, A. (2012) in Lipases and Phospholipases (Sandoval, G., ed.) Humana Press, pp. 3-30, doi: 10.1007/978-1-61779-600-5_1.

  2. 2

    Gaur, R., Hemamalini, R., and Khare, S. (2017) in Current Developments in Biotechnology and Bioengineering (Pandey, A. N. S., and Soccol, C. R., eds.) Elsevier, pp. 175-198, doi: 10.1016/B978-0-444-63662-1.00008-7.

  3. 3

    Romano, D., Bonomi, F., Mattos, M. C., Fonseca, T. D., Oliveira, M. D. F., and Molinari, F. (2015) Esterases as stereoselective biocatalysts, Biotechnol. Adv., 33, 547-565, doi: 10.1016/j.biotechadv.2015.01.006.

    CAS  Article  Google Scholar 

  4. 4

    Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., and Schrag, J. (1992) The α/β hydrolase fold, Protein Eng., 5, 197-211, doi: 10.1093/protein/5.3.197.

    CAS  Article  Google Scholar 

  5. 5

    Nardini, M., and Dijkstra, B. W. (1999) α/β Hydrolase fold enzymes: the family keeps growing, Curr. Opin. Struct. Biol., 9, 732-737, doi: 10.1016/S0959-440X(99)00037-8.

    CAS  Article  Google Scholar 

  6. 6

    Arpigny, J., and Jaeger, K. (1999) Bacterial lipolytic enzymes: classification and properties, Biochem. J., 343, 177-183, doi: 10.1042/bj3430177.

    CAS  Article  Google Scholar 

  7. 7

    Ferrer, M., Bargiela, R., Martínez-Martínez, M., Mir, J., Koch, R., Golyshina, O. V., and Golyshin, P. N. (2015) Biodiversity for biocatalysis: a review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes, Biocat. Biotrans., 33, 235-249, doi: 10.3109/10242422.2016.1151416.

    CAS  Article  Google Scholar 

  8. 8

    Kim, T. D. (2017) Bacterial hormone-sensitive lipases (bHSLs): emerging enzymes for biotechnological applications, J. Microbiol. Biotechnol., 27, 1907-1915, doi: 10.4014/jmb.1708.08004.

    CAS  Article  Google Scholar 

  9. 9

    Mirete, S., Morgante, V., and González-Pastor, J. E. (2016) Functional metagenomics of extreme environments, Curr. Opin. Biotechnol., 38, 143-149, doi: 10.1016/j.copbio.2016.01.017.

    CAS  Article  Google Scholar 

  10. 10

    Handelsman, J. (2004) Metagenomics: application of genomics to uncultured microorganisms, Microbiol. Mol. Biol. Rev., 68, 669-685, doi: 10.1128/MMBR.68.4.669-685.2004.

    CAS  Article  Google Scholar 

  11. 11

    López-López, O., Cerdán, M. E., and Siso, M. I. (2014) New extremophilic lipases and esterases from metagenomics, Curr. Prot. Pept. Sci., 15, 445-455, doi: 10.2174/1389203715666140228153801.

    Article  Google Scholar 

  12. 12

    Petrovskaya, L. E., Novototskaya-Vlasova, K. A., Spirina, E. V., Durdenko, E. V., Lomakina, G. Y., Zavialova, M. G., Nikolaev, E. N., and Rivkina, E. M. (2016) Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library, FEMS Microbiol. Ecol., 92, fiw046, doi: 10.1093/femsec/fiw046.

    Article  Google Scholar 

  13. 13

    Petrovskaya, L. E., Novototskaya-Vlasova, K. A., Gapizov, S. S., Spirina, E. V., Durdenko, E. V., and Rivkina, E. M. (2017) New member of the hormone-sensitive lipase family from the permafrost microbial community, Bioengineered, 8, 420-423, doi: 10.1080/21655979.2016.1230571.

    CAS  Article  Google Scholar 

  14. 14

    Boyko, K. M., Kryukova, M. V., Petrovskaya, L. E., Nikolaeva, A. Y., Korzhenevsky, D. A., Novototskaya-Vlasova, K. A., Rivkina, E. M., Dolgikh, D. A., Kirpichnikov, M. P., and Popov, V. O. (2020) Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine, PLoS One, 15, e0226838, doi: 10.1371/journal.pone.0226838.

    Article  Google Scholar 

  15. 15

    Madeira, F., Park, Y. M., Lee, J., Buso, N., Gur, T., Madhusoodanan, N., Basutkar, P., Tivey, A. R. N., Potter, S. C., Finn, R. D., and Lopez, R. (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019, Nucleic Acids Res., 47, W636-W641, doi: 10.1093/nar/gkz268.

    CAS  Article  Google Scholar 

  16. 16

    Alcaide, M., Stogios, P. J., Lafraya, Á., Tchigvintsev, A., Flick, R., Bargiela, R., Chernikova, T. N., Reva, O. N., Hai, T., Leggewie, C. C., Katzke, N., La Cono, V., Matesanz, R., Jebbar, M., Jaeger, K.-E., Yakimov, M. M., Yakunin, A. F., Golyshin, P. N., Golyshina, O. V., Savchenko, A., Ferrer, M., and MAMBA Consortium (2015) Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats, Environ. Microbiol., 17, 332-345, doi: 10.1111/1462-2920.12660.

    CAS  Article  Google Scholar 

  17. 17

    Li, P. Y., Ji, P., Li, C. Y., Zhang, Y., Wang, G. L., Zhang, X. Y., Xie, B. B., Qin, Q. L., Chen, X. L., Zhou, B. C., and Zhang, Y. Z. (2014) Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family, J. Biol. Chem., 289, 19031-19041, doi: 10.1074/jbc.M114.574913.

    CAS  Article  Google Scholar 

  18. 18

    Kourist, R., Brundiek, H., and Bornscheuer, U. T. (2010) Protein engineering and discovery of lipases, Eur. J. Lipid Sci. Technol., 112, 64-74, doi: 10.1002/ejlt.200900143.

    CAS  Article  Google Scholar 

  19. 19

    Jochens, H., Hesseler, M., Stiba, K., Padhi, S. K., Kazlauskas, R. J., and Bornscheuer, U. T. (2011) Protein engineering of alpha/beta-hydrolase fold enzymes, Chembiochem, 12, 1508-1517, doi: 10.1002/cbic.201000771.

    CAS  Article  Google Scholar 

  20. 20

    Kulakova, L., Galkin, A., Nakayama, T., Nishino, T., and Esaki, N. (2004) Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability, Biochim. Biophys. Acta, 1696, 59-65, doi: 10.1016/j.bbapap.2003.09.008.

    CAS  Article  Google Scholar 

  21. 21

    Kobayashi, R., Hirano, N., Kanaya, S., Saito, I., and Haruki, M. (2010) Enhancement of the enzymatic activity of Escherichia coli acetyl esterase by random mutagenesis, J. Mol. Cat. B Enzymatic, 67, 155-161, doi: 10.1016/j.molcatb.2010.08.003.

    CAS  Article  Google Scholar 

  22. 22

    Manco, G., Mandrich, L., and Rossi, M. (2001) Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature, J. Biol. Chem., 276, 37482-37490, doi: 10.1074/jbc.M103017200.

    CAS  Article  Google Scholar 

  23. 23

    Sayer, C., Isupov, M. N., Bonch-Osmolovskaya, E., and Littlechild, J. A. (2015) Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis, FEBS J., 282, 2846-2857, doi: 10.1111/febs.13326.

    CAS  Article  Google Scholar 

  24. 24

    Kim, B. Y., Yoo, W., Huong Luu Le, L. T., Kim, K. K., Kim, H. W., Lee, J. H., Kim, Y. O., and Kim, T. D. (2019) Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4, Arch. Biochem. Biophys., 663, 132-142, doi: 10.1016/j.abb.2019.01.010.

    CAS  Article  Google Scholar 

  25. 25

    Lan, D., Xu, H., Xu, J., Dubin, G., Liu, J., Khan, F. I., and Wang, Y. (2017) Malassezia globosa MgMDL2 lipase: crystal structure and rational modification of substrate specificity, Biochem. Biophys. Res. Commun., 488, 259-265, doi: 10.1016/j.bbrc.2017.04.103.

    CAS  Article  Google Scholar 

  26. 26

    Jeon, J. H., Lee, H. S., Kim, J. T., Kim, S. J., Choi, S. H., Kang, S. G., and Lee, J. H. (2012) Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment, Appl. Microbiol. Biotechnol., 93, 623-631, doi: 10.1007/s00253-011-3433-x.

    CAS  Article  Google Scholar 

  27. 27

    Novototskaya-Vlasova, K., Petrovskaya, L., Yakimov, S., and Gilichinsky, D. (2012) Cloning, purification, and characterization of a cold adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg, FEMS Microbiol. Ecol., 82, 367-375, doi: 10.1111/j.1574-6941.2012.01385.x.

    CAS  Article  Google Scholar 

  28. 28

    Novototskaya-Vlasova, K., Petrovskaya, L., Kryukova, E., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. (2013) Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis K5T, Protein Expr. Purif., 91, 96-103, doi: 10.1016/j.pep.2013.07.011.

    CAS  Article  Google Scholar 

  29. 29

    Novototskaya-Vlasova, K., Petrovskaya, L., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. (2013) Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants, Biochemistry (Moscow), 78, 385-394, doi: 10.1134/S000629791304007X.

    CAS  Article  Google Scholar 

  30. 30

    Siddiqui, K. S., and Cavicchioli, R. (2006) Cold-adapted enzymes, Annu. Rev. Biochem., 75, 403-433, doi: 10.1146/annurev.biochem.75.103004.142723.

    CAS  Article  Google Scholar 

  31. 31

    Feller, G., and Gerday, C. (2003) Psychrophilic enzymes: hot topics in cold adaptation, Nat. Rev. Microbiol., 1, 200-208, doi: 10.1038/nrmicro773.

    CAS  Article  Google Scholar 

  32. 32

    Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr. D Biol. Crystallogr., 60, 2126-2132, doi: 10.1107/S0907444904019158.

    Article  Google Scholar 

Download references

Funding

This work was partially supported by the Russian Foundation for Basic Research (project No. 18-04-00491) and by the Molecular and Cellular Biology Program of the Russian Academy of Sciences.

Author information

Affiliations

Authors

Corresponding author

Correspondence to L. E. Petrovskaya.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare no conflict of interest in financial or any other sphere.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kryukova, M., Petrovskaya, L., Novototskaya-Vlasova, K. et al. Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties. Biochemistry Moscow 85, 709–716 (2020). https://doi.org/10.1134/S0006297920060085

Download citation

Keywords

  • PMGL2 esterase
  • HSL family
  • GCSAG motif
  • mutagenesis
  • thermal stability
  • three-dimensional structure