Email this article О-Аcetylhomoserine sulfhydrylase from Clostridioides difficile: the role of tyrosine residues of the active center
- Authors: Kulikova V.V1, Revtovich S.V1, Lyfenko A.D1, Morozova E.A1, Koval V.S1, Bazhulina N.P1, Demidkina T.V1
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Affiliations:
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
- Issue: Vol 88, No 5 (2023)
- Pages: 737-747
- Section: Articles
- URL: https://journals.rcsi.science/0320-9725/article/view/144669
- DOI: https://doi.org/10.31857/S0320972523050032
- EDN: https://elibrary.ru/AXKFQQ
- ID: 144669
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Abstract
O-acetylhomoserine sulfhydrylase is one of the key enzymes in the biosynthesis of methionine in Clostridioides difficile. The mechanism of the γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among pyridoxal-5′-phosphate-dependent enzymes involved in the metabolism of cysteine and methionine. To clarify the role of the active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements for phenylalanine and alanine were obtained. The catalytic and spectral properties of mutant forms were investigated. The rate of the γ-substitution reaction catalyzed by mutant forms with the replacement of the Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the residues Tyr52 and Tyr107 led to a decrease in the affinity of the apoenzyme to the 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 the 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 can act as a general acid catalyst at the stage of acetate elimination.
About the authors
V. V Kulikova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
S. V Revtovich
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
A. D Lyfenko
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
E. A Morozova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
V. S Koval
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
N. P Bazhulina
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
T. V Demidkina
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: vitviku@yandex.ru
119991 Moscow, Russia
References
- 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, doi: 10.1016/j.pep.2020.105810.
- 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, doi: 10.1002/iub.2139.
- 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, doi: 10.1016/S0021-9258(18)62537-2.
- Yamagata, S. (1971) Homocysteine synthesis in yeast. Partial purification and properties of O-acetylhomoserine sulfhydrylase, J. Biochem., 70, 1035-1045, doi: 10.1093/oxfordjournals.jbchem.a129712.
- 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, doi: 10.1128/jb.130.1.62-73.1977.
- 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, doi: 10.1007/s00253-003-1306-7.
- 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, doi: 10.1099/13500872-141-2-431.
- Belfaiza, J., Martel, A., Margarita, D., and Saint Girons, I. (1998) Direct sulfhydrylation for methionine biosynthesis in Leptospira meyeri, J. Bacteriol., 180, 250-255, doi: 10.1128/jb.180.2.250-255.
- 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, doi: 10.1016/s0167-4838(01)00245-x.
- Krishnamoorthy, K., and Begley, T.P. (2011) Protein thiocarboxylate-dependent methionine biosynthesis in Wolinella succinogenes, J. Am. Chem. Soc., 133, 379-386, doi: 10.1021/ja107424t.
- 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, doi: 10.1107/S0907444911028010.
- 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, doi: 10.1016/j.jbc.2021.100797.
- 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, doi: 10.1515/BC.2003.043.
- 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, doi: 10.1271/bbb.64.2336.
- 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, doi: 10.1016/j.biochi.2014.01.007.
- 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, doi: 10.1016/j.bbapap.2014.12.027.
- 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, doi: 10.1016/0003-2697(76)90527-3.
- Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680-685, doi: 10.1038/227680a0.
- Kredich, N. M., and Becker, M. A. (1971) Cysteine biosynthesis: serine transacetylase and O-acetylserine sulfhydrylase, Methods Enzymol., 17 B, 459-470, doi: 10.1016/0076-6879(71)17082-6.
- Peterson, E. A., and Sober, H. A. (1954) Preparation of crystalline phosphorylated derivatives of vitamin B6, J. Am. Chem. Soc., 76, 169-175, doi: 10.1021/ja01630a045.
- 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, doi: 10.1046/j.1432-1327.2000.01185.x.
- Scatchard, G. (1949) The attraction of proteins for small molecules and ions, Ann. NY Acad. Sci., 51, 660-672, doi: 10.1111/j.1749-6632.1949.tb27297.x.
- 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, doi: 10.1006/jmbi.1999.2997.
- 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, doi: 10.1021/bi00454a020.
- 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, doi: 10.1006/jmbi.2001.4880.
- 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, doi: 10.1038/314235a0.
- 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, doi: 10.1021/bi00140a025.
- 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, doi: 10.1016/j.bbapap.2006.05.001.
- 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, doi: 10.1002/prot.24707.
