Skip to main content
SpringerLink
Log in

O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5′-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.

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.
Fig. 5.
Fig. 6.

Similar content being viewed by others

Abbreviations

OAH:

O-acetyl-L-homoserine

OAHS:

O-acetylhomoserine sulfhydrylase

PLP:

pyridoxal-5′-phosphate

References

  1. Kulikova, V. V., Anufrieva, N. V., Kotlov, M. I., Morozova, E. A., Koval, V. S., Belyi, Y. F., Revtovich, S. V., and Demidkina, T. V. (2021) O-acetylhomoserine sulfhydrylase from Clostridium novyi. Cloning, expression of the gene and characterization of the enzyme, Protein Expr. Purif., 180, 105810, https://doi.org/10.1016/j.pep.2020.105810.

    Article  CAS  PubMed  Google Scholar 

  2. Kulikova, V. V., Revtovich, S. V., Bazhulina, N. P., Anufrieva, N. V., Kotlov, M. I., Koval, V. S., Morozova, E. A., Hayashi, H., Belyi, Y. F., and Demidkina, T. V. (2019) Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations, IUBMB Life, 71, 1815-1823, https://doi.org/10.1002/iub.2139.

    Article  CAS  PubMed  Google Scholar 

  3. Kerr, D. S. (1971) O-Acetylhomoserine sulfhydrylase from Neurospora. Purification and consideration of its function in homocysteine and methionine synthesis, J. Biol. Chem., 246, 95-102, https://doi.org/10.1016/S0021-9258(18)62537-2.

    Article  CAS  PubMed  Google Scholar 

  4. Yamagata, S. (1971) Homocysteine synthesis in yeast. Partial purification and properties of O-acetylhomoserine sulfhydrylase, J. Biochem., 70, 1035-1045, https://doi.org/10.1093/oxfordjournals.jbchem.a129712.

    Article  CAS  PubMed  Google Scholar 

  5. Murooka, Y., Kakihara, K., Miwa, T., Seto, K., and Harada, T. (1977) O-alkylhomoserine synthesis catalyzed by O-acetylhomoserine sulfhydrylase in microorganisms, J. Bacteriol., 130, 62-73, https://doi.org/10.1128/jb.130.1.62-73.1977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lee, H., and Hwang, B. (2003) Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation, Appl. Microbiol. Biotechnol., 62, 459-467, https://doi.org/10.1007/s00253-003-1306-7.

    Article  CAS  PubMed  Google Scholar 

  7. Foglino, M., Borne, F., Bally, M., Ball, G., and Patte, J. (1995) A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa, Microbiology, 141, 431-439, https://doi.org/10.1099/13500872-141-2-431.

    Article  CAS  PubMed  Google Scholar 

  8. Belfaiza, J., Martel, A., Margarita, D., and Saint Girons, I. (1998) Direct sulfhydrylation for methionine biosynthesis in Leptospira meyeri, J. Bacteriol., 180, 250-255, https://doi.org/10.1128/jb.180.2.250-255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shimizu, H., Yamagata, S., Masui, R., Inoue, Y., Shibata, T., Yokoyama, S., Kuramitsu, S., and Iwama, T. (2001) Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product, Biochim. Biophys. Acta, 1549, 61-72, https://doi.org/10.1016/s0167-4838(01)00245-x.

    Article  CAS  PubMed  Google Scholar 

  10. Krishnamoorthy, K., and Begley, T. P. (2011) Protein thiocarboxylate-dependent methionine biosynthesis in Wolinella succinogenes, J. Am. Chem. Soc., 133, 379-386, https://doi.org/10.1021/ja107424t.

    Article  CAS  PubMed  Google Scholar 

  11. Tran, T. H., Krishnamoorthy, K., Begley, T. P., and Ealick, S. E. (2011) A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes, Acta Cryst., D67, 831-838, https://doi.org/10.1107/S0907444911028010.

    Article  CAS  Google Scholar 

  12. Brewster, J. L., Pachl, P., McKellar, J. L., Selmer, M., Squire, C. J., and Patrick, W. M. (2021) Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis, J. Biol. Chem., 296, 100797, https://doi.org/10.1016/j.jbc.2021.100797.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Messerschmidt, A., Worbs, M., Steegborn, C., Wahl, M. C., Huber, R., Laber, B., and Clausen, T. (2003) Determinants of enzymatic specificity in the Cys-Met-metabolism PLP-dependent enzymes family: crystal structure of cystathionine γ-lyase from yeast and intrafamiliar structure comparison, Biol. Chem., 384, 373-386, https://doi.org/10.1515/BC.2003.043.

    Article  CAS  PubMed  Google Scholar 

  14. Inoue, H., Inagaki, K., Adachi, N., Tamura, T., Esaki, N., Soda, K., and Tanaka, H. (2000) Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida, Biosci. Biotechnol. Biochem., 64, 2336-2343, https://doi.org/10.1271/bbb.64.2336.

    Article  CAS  PubMed  Google Scholar 

  15. Revtovich, S. V., Faleev, N. G., Morozova, E. A., Anufrieva, N. V., Nikulin, A. D., and Demidkina, T. V. (2014) Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and β-protons of competitive inhibitors, Biochimie, 101, 161-167, https://doi.org/10.1016/j.biochi.2014.01.007.

    Article  CAS  PubMed  Google Scholar 

  16. Anufrieva, N. V., Faleev, N. G., Morozova, E. A., Bazhulina, N. P., Revtovich, S. V., Timofeev, V. P., Tkachev, Y. V., Nikulin, A. D., and Demidkina, T. V. (2015) The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase, Biochim. Biophys. Acta, 1854, 1220-1228, https://doi.org/10.1016/j.bbapap.2014.12.027.

    Article  CAS  PubMed  Google Scholar 

  17. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254, https://doi.org/10.1016/0003-2697(76)90527-3.

    Article  CAS  PubMed  Google Scholar 

  18. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680-685, https://doi.org/10.1038/227680a0.

    Article  CAS  PubMed  Google Scholar 

  19. Kredich, N. M., and Becker, M. A. (1971) Cysteine biosynthesis: serine transacetylase and O-acetylserine sulfhydrylase (Salmonella typhimurium), Methods Enzymol., 17 B, 459-470, https://doi.org/10.1016/0076-6879(71)17082-6.

    Article  Google Scholar 

  20. Peterson, E. A., and Sober, H. A. (1954) Preparation of crystalline phosphorylated derivatives of vitamin B6, J. Am. Chem. Soc., 76, 169-175, https://doi.org/10.1021/ja01630a045.

    Article  CAS  Google Scholar 

  21. Bazhulina, N. P., Morozov, Y. V., Papisova, A. I., and Demidkina, T. V. (2000) Pyridoxal 5′-phoshate schiff base in Citrobacter freundii tyrosine phenol-lyase. Ionic and tautomeric equilibria, Eur. J. Biochem., 267, 1830-1836, https://doi.org/10.1046/j.1432-1327.2000.01185.x.

    Article  CAS  PubMed  Google Scholar 

  22. Scatchard, G. (1949) The attraction of proteins for small molecules and ions, Ann. NY Acad. Sci., 51, 660-672, https://doi.org/10.1111/j.1749-6632.1949.tb27297.x.

    Article  CAS  Google Scholar 

  23. Käck, H., Sandmark, J., Gibson, K., Schneider, G., and Lindqvist, Y. (1999) Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes, J. Mol. Biol., 291, 857-876, https://doi.org/10.1006/jmbi.1999.2997.

    Article  PubMed  Google Scholar 

  24. Brzović, P., Holbrook, E. L., Greene, R. C., and Dunn, M. F. (1990) Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions, Biochemistry, 29, 442-451, https://doi.org/10.1021/bi00454a020.

    Article  PubMed  Google Scholar 

  25. Steegborn, C., Laber, B., Messerschmidt, A., Huber, R., and Clausen, T. (2001) Crystal structures of cystathionine γ-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor, J. Mol. Biol., 311, 789-801, https://doi.org/10.1006/jmbi.2001.4880.

    Article  CAS  PubMed  Google Scholar 

  26. Fersht, A. R., Shi, J. P., Knill-Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Carter, P., Waye, M. M., and Winter, G. (1985) Hydrogen bonding and biological specificity analysed by protein engineering, Nature, 314, 235-238, https://doi.org/10.1038/314235a0.

    Article  CAS  PubMed  Google Scholar 

  27. Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., and Kagamiyama, H. (1992) Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5′-phosphate, Biochemistry, 31, 5878-5887, https://doi.org/10.1021/bi00140a025.

    Article  CAS  PubMed  Google Scholar 

  28. Demidkina, T. V., Faleev, N.G., Papisova, A. I., Bazhulina, N. P., Kulikova, V. V., Gollnick, P. D., and Phillips, R. S. (2006) Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C–H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site, Biochim. Biophys. Acta, 1764, 1268-1276, https://doi.org/10.1016/j.bbapap.2006.05.001.

    Article  CAS  PubMed  Google Scholar 

  29. Astegno, A., Allegrini, A., Piccoli, S., Giorgetti, A., and Dominici, P. (2015) Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase, Proteins, 83, 78-90, https://doi.org/10.1002/prot.24707.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported by the Russian Science Foundation (project no. 22-24-00255).

Author information

Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, Moscow, Russia

    Vitalia V. Kulikova, Svetlana V. Revtovich, Anna D. Lyfenko, Elena A. Morozova, Vasiliy S. Koval, Natalya P. Bazhulina & Tatyana V. Demidkina

Authors
  1. Vitalia V. Kulikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Svetlana V. Revtovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna D. Lyfenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elena A. Morozova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vasiliy S. Koval
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Natalya P. Bazhulina
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tatyana V. Demidkina
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.V. Kulikova performed kinetic and spectral studies, wrote an article, S.V. Revtovich isolated enzymes, A.D. Lyfenko grew biomass, E.A. Morozova performed mutagenesis, V.S. Koval performed studies of the isotope exchange reaction, N.P. Bazhulina analyzed spectra by the method of log-normal decomposition, T.V. Demidkina coordinated the overall work.

Corresponding author

Correspondence to Vitalia V. Kulikova.

Ethics declarations

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kulikova, V.V., Revtovich, S.V., Lyfenko, A.D. et al. O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site. Biochemistry Moscow 88, 600–609 (2023). https://doi.org/10.1134/S0006297923050036

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297923050036

Keywords

Search

Navigation