Department of Protein Synthesis Enzymology


Mykhailo A. Tukalo

Professor, Dr. Sci. (Mol. Biol.),
Corresponding Member of NASU,
Deputy director for scientific recearch
Tel: (380-44) 200-03-35
Fax (380-44) 526-07-59

Group of Molecular Pharmacology

Head: Zenoviy Yu. Tkachuk
Ph.D. (Mol. Biol.), Senior Staff Scientist

Group of system biology

Head: Group of system biology
Professor, Dr. Sci. (Mol. Biol.)

Education and Degrees:

19681973 Graduate Student, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine, M.Sc. (biochemistry)

1981 Ph.D. (molecular biology)

1989 Dr.Sci. (molecular biology)

2005 Professor, Taras Shevchenko National University of Kyiv

2009 Corresponding Member of NASU

Professional Employment:

1973 Research Assistant, Institute of Molecular Biology and Genetics (IMBG), NASU, Kyiv, Ukraine (molecular biology)

19741977 Visiting Scientist at the Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia

19771982 Junior Research Scientist, Group leader, IMBG NASU, Kyiv, Ukraine

19821986 Senior Research Scientist, Group leader, IMBG NASU, Kyiv, Ukraine

19861990 Leading Research Scientist, Group leader, IMBG NASU, Kyiv, Ukraine

since 1990 Head of the Department of Protein Synthesis Enzymology, IMBG NASU, Kyiv, Ukraine

19942002 Staff Scientist at the European Molecular Biology Laboratory, Grenoble Outstation, France

since 2006 Deputy Director in Scientific Work, IMBG NASU, Kyiv, Ukraine

Honours, Prizes, Awards:

1982 Medal In memory of the 1500 anniversary of the city of Kyiv

1986 State Prize of Ukraine in Science and Technology for contribution to molecular biology through research on the regulation of protein biosynthesis

19952000 Howard Hughes Medical Institute International Research Grant INTAS Collaborative Grant

2011 Diploma of Verhovna Rada of Ukraine

2011 Gershenson Award of National Academy of Sciences of Ukraine

Research Area:

Study on the molecular basis of decoding genetic information, translation quality control and RNA-protein recognition

urrent Research Activities and Recent Achievements:

Structural and functional study on aminoacyl-tRNA synthetases.

The research of the last years was directed at the identification of the structural bases of the decoding of genetic information. The current area is the specific recognition of aminoacyl-tRNA synthetases (aaRSs) for their cognate amino acid and tRNA, mechanisms of catalysis and editing. Using biochemical methods, site-directed mutagenesis and X-ray crystallography, a work was carried out on different prokaryotic (including important pathogenic bacteria Enterococcus faecalis, Mycobacterium tuberculosis and Streptococcus pneumoniae), eukaryotic and archaeal systems. In collaboration with Dr. S. Cusack (the European Molecular Biological Laboratory) the 3-dimensional structures of several aaRSs and their complexes with various combinations of substrata, including the tRNA complexes, are being studied. The synthesis of specific products by aaRSs has been shown to be accompanied by the conformational changes both in the active centre of enzyme and beyond.The data obtained allowed the understanding of the mechanism of amino acids activation and the molecular mechanism of the recognition of homologous tRNA and their aminoacylation by these enzymes.

Fig. 1. Conformational changes occuring when a histidine binds in the active site of HisRSTT (the left) or tyrosyl-adenylate analogue binds in the active site of TyrRSTT (the right)
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases.

In prokaryotic cells three tRNA species, tRNASer, tRNATyr and tRNALeu, possess a long variable arm of 1120 nucleotides (a type 2 tRNAs) rather than the usual 4 or 6 ( a type 1 tRNAs). We studied the molecular basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl- tRNA synthetases (SerRS, TyrRS and LeuRS) using X-ray crystallography and chemical probing of tRNA in solution. The determination of a complex of SerRs and tRNASer provided the first information on the structure of a tRNA with a long variable arm and elucidated the details of how enzyme interacts with tRNA. As a result of solving the structure of TyrRS an unusual for these enzymes type recognition of tRNA has been demonstrated for the first time. Tyrosyl-tRNA synthetase belongs to the first structural class, but its type of recognition is specific for the class 2 aaRSs, where tRNA interacts with an enzyme from the side of a long variable arm. Finally, after the structure of a complex of the leucyl-tRNA synthetase with tRNALeu was determined, a full picture of the interaction of the synthetases with tRNA, which has a long variable arm, has been obtained. The distinctions, revealed in the tertiary structures of tRNASer, tRNATyr and tRNALeu, using the methods of X-ray structure analysis and chemical modification, allowed us to understand the importance of their role in the recognition and discrimination by homologous aaRSs.

Fig. 2. Cloverleaf structures of T. thermophilus tRNATyr, tRNASer and tRNALeu with position of phosphates protected by cognate synthetase from alkylation with ethylnitrosourea
Fig. 3. Recognition of tRNAs with a long variable arm by cognate aminoacyl-tRNA synthetases
The molecular basis for editing errors by aminoacyl-tRNA syntetases.

The affinity difference is not enough for the aminoacyltRNA synthetases to discriminate strictly between similar amino acids. When a tRNA is acylated with the wrong amino acid this would lead to an error in the incorporation of genetically coded amino acids into protein. To overcome this problem, several aaRSs have developed the ability to hydrolyze the mischarged tRNA in an extra editing domain. We are studying the molecular mechanisms of editing by synthetases from two different classes: Thermus thermophilus leucyltRNA leucyltRNA synthetase (LeuRSTT) from class I and Enterococcus fecalis prolyl-tRNA synthetase (ProRSEF) from class II. To understand the mechanisms of editing reaction for enzymes with absolutely different architecture of editing domains, we have used a number of approaches, including molecular modeling, quantum-mechanical calculations, site-directed mutagenesis and enzyme modification of tRNA. Our intensive alanine scanning mutagenesis of LeuRSTT and ProRSEF editing sites has failed to identify catalytic residues for hydrolysis within the active site. On the other hand, modification of tRNAPro at the 2-OH of A76 and tRNALeu at the 3-OH of A76 by replacing each of them with a hydrogen or fluorine, revealed an essential function of these groups in hydrolysis. On the basis of obtained experimental results and our QM/ MM calculations we suggest a tRNA-assisted mechanism of post-transfer editing by LeuRS and ProRS in which 2- or 3-OH group of the substrate plays a key role.

Fig. 4. tRNA-assisted mechanism of post-transfer editing by leucyltRNA synthetase
Development of novel classes of antibiotics.

We are using the differences between human and prokaryotic prolyl-, tyrosyl- and leucyl-tRNA synthetases for the development of the inhibitors as potential drugs against Mycobacterium tuberculosis, Enterococcus faecalis and Streptococcus pneumonia. The search strategy for antibacterial compounds is based on the combination of X-ray structural analysis of the target protein, computer modelling of the interaction of low-molecular ligands with the target protein and synthetic procedures of combinatorial chemistry.

Fig. 5. Exploitation of structural differences of human and pathogenic bacterial prolyl-tRNA synthetases for the identification of novel inhibitors as potential anti-pathogen drugs

National Grants:

Projects of National Academy of Sciences of Ukraine:

  • 20122016 Progect: Target-directed search for a new antibacterial, antiviral and antitumor agents (scientific supervisor M. Tukalo)
  • 20102014 N 30/10 Progect: Development of targetspecific technologies for searching of the aminoacyltRNA synthetases inhibitors with selective action against causative agents of human infection diseases (scientific supervisor M. Tukalo)

Projects of State Agency on Science, Innovations and Informatization of Ukraine:

  • 20122015 N 059/514 Project: Development of the methodologies target-directed, rational search for new anti- TB drugs (scientific supervisor M. Tukalo)

International Grants:

  • 20132016 GDRI (International Research Networks) Project: From molecular to cellular events in human pathologies (scientific supervisors Prof. P. Curmi and Prof. O. Lavrik )
  • 20112014 7th Framework Programme (FP7) FP7- INCO-2011-6, ERA-WIDE Project: Strengthening cooperation in Molecular Biomedicine between EU and UKRAINE, COMBIOM (scientific supervisor Prof. A. Elskaya)
  • 20112012 STCU (Science and Technology Center in Ukraine) Research Grants
  • 20072009 STCU Research Grants
  • 19921995 NATO (North Atlantic Treaty Organization) Collaborative Research Grant


with Ukrainian organizations:

  • Taras Shevchenko National University of Kyiv (Kyiv)
  • Bogomoletz Institute of Physiology, NASU (Kyiv)

with foreign organizations:

  • European Molecular Biology Laboratory (Grenoble, France)
  • Institute Gustave-Roussy (Paris, France)
  • Laboratory of Enzymology and Structural Biochemistry, CNRS (Gif-sur-Yvette, France)
  • International Institute of Molecular and Cell Biology (Warsaw, Poland)
  • Institute of Chemical Biology and Fundamental Medicine, SB RAS (Novosibirsk, Russia)

Selected publications:

  1. Gudzera O. I., Golub A.G., Bdzhola V. G., Volynets G. P., Lukashov S.S., Kovalenko O. P., Kriklivyi I. A., Yaremchuk A.D., Starosyla S. A., Yarmoluk S. M., Tukalo M.A. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase. Bioorganic & Medicinal Chemistry. 2016, 24: 1023-131.
  2. Gudzera O. I., Golub A. G., Bdzhola V. G., Volynets G. P., Kovalenko O. P., Boyarshin K. S., Yaremchuk A.D., Protopopov M.V., Yarmoluk S. M., Tukalo M. A. Identification of Mycobacterium tuberculosis leucyl-tRNA synthetase (LeuRS) inhibitors among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one. Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1190712.
  3. Boyarshin K., Priss A., Rayevskiy A, Ilchenko M, Dubey I, Kriklivyi I, Yaremchuk A. Tukalo M. A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: Catalysis of hydrolytic reaction by prolyl-tRNA synthetase of bacterial type. Journal of Biomolecular Structure and Dynamics. DOI: 10.1080/07391102.2016.1155171.
  4. Rayevsky A. V., Tukalo M. A. Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates. Biopolymers and Cell. 2016, 32(1):6169.
  5. Rybak MYu, Kovalenko OP, Kriklivyi IA, Tukalo MA. Cloning, expression and purification of D-Tyr-tRNATyr deacylase from Thermus thermophilus. Biopolymers & Cell, 2015;31(3):179186.
  6. Boyarshin K, Priss A, Rayevskiy A, Ilchenko M, Dubey I, Kriklivyi I, Yaremchuk A, Tukalo M. A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: Catalysis of hydrolytic reaction by prolyl-tRNA synthetase of bacterial type. Journal of Biomolecular Structure & Dynamics. In Press.
  7. Gudzera OI, Golub AG, Bdzhola VG, Volynets GP, Lukashov SS, Kovalenko OP, Kriklivyi IA, Yaremchuk AD, Starosyla SA, Yarmoluk SM, Tukalo MA. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase. Bioorganic & Medicinal Chemistry. In Press.
  8. Kovalenko O, Gudzera O, Tukalo M. Mutagenesis probe into posttransfer editing mechanism of prokaryotic leucyl-tRNA synthetase. The FEBS Letters. In Press.
  9. Crepin T, Shalak VF, Yaremchuk AD, et al. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes. Nucleic Acids Research. 2014;1:1-10.
  10. Demianenko E, Ilchenko M, Grebenyuk A, et al. A theoretical study on ascorbic acid dissociation in water clusters. J. Mol. Model. 2014;20(3):1-8.
  11. Tukalo M. A., Yaremchuk G. D., Kovalenko O. P., Kriklivyi I. A., Gudzera O. I. Recognition of tRNAs with a long variable arm by aminoacyl- tRNA synthetases Biopolym. Cell. 2013; 29(4):311-323doi:10.7124/bc.000825
  12. Yaremchuk AD, Kovalenko OP, Gudzera OI, Tukalo MA. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase Biopolym. Cell. 2011; 27(6): 43641.doi: 10.7124/bc.000114
  13. Rock FL, Mao W, Yaremchuk A, et al. An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science. 2007;316(5832):175961. doi: 10.1126/science.1142189
  14. Tukalo M, Yaremchuk A, Fukunaga R, Yokoyama S, Cusack S. The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation. Nat Struct Mol Biol. 2005;12(10):92330.doi:10.1038/nsmb986
  15. Lincecum TL Jr, Tukalo M, Yaremchuk A, et al. Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase. Mol Cell. 2003;11(4):95163.doi:10.1016/S1097-2765(03)00098-4
  16. Yaremchuk A, Kriklivyi I, Tukalo M, Cusack S. Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition. EMBO J. 2002;21(14):382940.doi: 10.1093/emboj/cdf373
  17. Yaremchuk A, Cusack S, Tukalo M. Crystal structure of a eukaryote/ archaeon-like protyl-tRNA synthetase and its complex with tRNAPro(CGG). EMBO J. 2000; 19(17): 474558.doi:10.1093/emboj/19.17.4745
  18. Cusack S, Yaremchuk A, Tukalo M. The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue. EMBO J. 2000;19(10):235161.doi:10.1093/emboj/19.10.2351
  19. Cusack S, Yaremchuk A, Tukalo M. The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site. EMBO J. 1996; 15(11):283442.
  20. Biou V, Yaremchuk A, Tukalo M, Cusack S. The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser). Science. 1994;263(5152):140410.doi:10.1126/science.8128220
  21. Belrhali H, Yaremchuk A, Tukalo M, et al. Crystal structures at2.5 angstrom resolution of seryl-tRNA synthetase complexedwith two analogs of seryl adenylate. Science. 1994; 263(5152):14326.doi:10.1126/science.8128224