Protein & Cell

, Volume 1, Issue 11, pp 1023–1032 | Cite as

Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

  • Prajwalini Mehere
  • Qian Han
  • Justin A. Lemkul
  • Christopher J. Vavricka
  • Howard Robinson
  • David R. Bevan
  • Jianyong LiEmail author
Research Article


Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.


tyrosine aminotransferase crystal structure substrate specificity tyrosine tyrosinemia 

Supplementary material

13238_2010_128_MOESM1_ESM.pdf (350 kb)
Supplementary material, approximately 350 KB.


  1. al-Hemidan, A.I., and al-Hazzaa, S.A. (1995). Richner-Hanhart syndrome (tyrosinemia type II). Case report and literature review. Ophthalmic Genet 16, 21–26.CrossRefGoogle Scholar
  2. Bein, N.N., and Goldsmith, H.S. (1977). Recurrent massive haemorrhage from benign hepatic tumours secondary to oral contraceptives. Br J Surg 64, 433–435.CrossRefGoogle Scholar
  3. Beneking, M., Schmidt, H., and Weiss, G. (1978). Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme. Eur J Biochem 82, 235–243.CrossRefGoogle Scholar
  4. Berendsen, H.J.C., Postma, J.P.M., Van Gunsteren, W.F., DiNola, A., and Haak, J.R. (1984). Molecular dynamics with coupling to an external bath. J Chem Phys 81, 3684–3690.CrossRefGoogle Scholar
  5. Berendsen, H.J.C., Postma, J.P.M., Van Gunsteren, W.F., and Hermans, J. (1981). Interaction models for water in relation to protein hydration. Jerusalem Symposia on Quantum Chemistry and Biochemistry 14, 331–342.CrossRefGoogle Scholar
  6. Blankenfeldt, W., Nowicki, C., Montemartini-Kalisz, M., Kalisz, H.M., and Hecht, H.J. (1999). Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode. Protein Sci 8, 2406–2417.CrossRefGoogle Scholar
  7. Buckley, W.T., and Milligan, L.P. (1978). Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates. Biochem J 176, 449–454.CrossRefGoogle Scholar
  8. Cavelier-Balloy, B., Venencie, P.Y., Lemonnier, V., Verola, O., Servant, J.M., Puissant, A., and Civatte, J. (1985). Histiocytoid hemangioma of the scalp. Ann Dermatol Venereol 112, 965–972.Google Scholar
  9. Charfeddine, C., Monastiri, K., Mokni, M., Laadjimi, A., Kaabachi, N., Perin, O., Nilges, M., Kassar, S., Keirallah, M., Guediche, M.N., et al. (2006). Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations. Mol Genet Metab 88, 184–191.CrossRefGoogle Scholar
  10. Ciechanover, A., Orian, A., and Schwartz, A.L. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22, 442–451.CrossRefGoogle Scholar
  11. Darden, T., York, D., and Pedersen, L. (1993). Particle mesh Ewald: an N.log(N) method for Ewald sums in large systems. J Chem Phys 98, 10089–10092.CrossRefGoogle Scholar
  12. DeLano, W.L. (2002). The PyMOL molecular graphics system. Delano Scientifc, San Carlos, CA, USA.Google Scholar
  13. Donini, S., Ferrari, M., Fedeli, C., Faini, M., Lamberto, I., Marletta, A. S., Mellini, L., Panini, M., Percudani, R., Pollegioni, L., et al. (2009). Recombinant production of eight human cytosolic aminotrans-ferases and assessment of their potential involvement in glyoxylate metabolism. Biochem J 422, 265–272.CrossRefGoogle Scholar
  14. Endo, F. (1998). Hereditary tyrosinemia type II. Ryoikibetsu Shokogun Shirizu, 134–136.Google Scholar
  15. Essmann, U., Perera, L., Berkowitz, M.L., Darden, T., Lee, H., and Pedersen, L.G. (1995). A smooth particle mesh Ewald method. J Chem Phys 103, 8577–8593.CrossRefGoogle Scholar
  16. Federici, G., Di Cola, D., Sacchetta, P., Di Ilio, C., Del Boccio, G., and Polidoro, G. (1978). Reversible inactivation of tyrosine aminotransferase from guinea pig liver by thiol and disulfide compounds. Biochem Biophys Res Commun 81, 650–655.CrossRefGoogle Scholar
  17. Fu, L., Dong, S.S., Xie, Y.W., Tai, L.S., Chen, L., Kong, K.L., Man, K., Xie, D., Li, Y., Cheng, Y., et al. (2010). Down-regulation of tyrosine aminotransferase at a frequently deleted region 16q22 contributes to the pathogenesis of hepatocellular carcinoma. Hepatology 51, 1624–1634.CrossRefGoogle Scholar
  18. Grishin, N.V., Phillips, M.A., and Goldsmith, E.J. (1995). Modeling of the spatial structure of eukaryotic ornithine decarboxylases. Protein Sci 4, 1291–1304.CrossRefGoogle Scholar
  19. Gross-Mesilaty, S., Hargrove, J.L., and Ciechanover, A. (1997). Degradation of tyrosine aminotransferase (TAT) via the ubiquitinproteasome pathway. FEBS Lett 405, 175–180.CrossRefGoogle Scholar
  20. Han, Q., Cai, T., Tagle, D.A., and Li, J. (2010). Structure, expression, and function of kynurenine aminotransferases in human and rodent brains. Cell Mol Life Sci 67, 353–368.CrossRefGoogle Scholar
  21. Han, Q., Fang, J., and Li, J. (2001). Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem J 360, 617–623.CrossRefGoogle Scholar
  22. Han, Q., Robinson, H., Cai, T., Tagle, D.A., and Li, J. (2009). Biochemical and structural properties of mouse kynurenine aminotransferase III. Mol Cell Biol 29, 784–793.CrossRefGoogle Scholar
  23. Hargrove, J.L., and Mackin, R.B. (1984). Organ specificity of glucocorticoid-sensitive tyrosine aminotransferase. Separation from aspartate aminotransferase isoenzymes. J Biol Chem 259, 386–393.Google Scholar
  24. Hess, B., Bekker, H., Berendsen, H.J.C., and Fraaije, J.G.E.M. (1997). LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18, 1463–1472.CrossRefGoogle Scholar
  25. Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008). GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J Chem Theory Comput 4, 435–447.CrossRefGoogle Scholar
  26. Holm, L., and Sander, C. (1993). Protein structure comparison by alignment of distance matrices. J Mol Biol 233, 123–138.CrossRefGoogle Scholar
  27. Hoover, W.G. (1985). Canonical dynamics: Equilibrium phase-space distributions. Phys Rev A 31, 1695–1697.CrossRefGoogle Scholar
  28. Huey, R., Morris, G.M., Olson, A.J., and Goodsell, D.S. (2007). A semiempirical free energy force field with charge-based desolvation. J Comput Chem 28, 1145–1152.CrossRefGoogle Scholar
  29. Jansonius, J.N. (1998). Structure, evolution and action of vitamin B6-dependent enzymes. Curr Opin Struct Biol 8, 759–769.CrossRefGoogle Scholar
  30. 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.CrossRefGoogle Scholar
  31. Ko, T.P., Wu, S.P., Yang, W.Z., Tsai, H., and Yuan, H.S. (1999). Crystallization and preliminary crystallographic analysis of the Escherichia coli tyrosine aminotransferase. Acta Crystallogr D Biol Crystallogr 55, 1474–1477.CrossRefGoogle Scholar
  32. Krissinel, E., and Henrick, K. (2004). Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr 60, 2256–2268.CrossRefGoogle Scholar
  33. Laskowski, R.A., Macarthur, M.W., Moss, D.S., and Thornton, J.M. (1993). Procheck—a program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283–291.CrossRefGoogle Scholar
  34. Macsai, M.S., Schwartz, T.L., Hinkle, D., Hummel, M.B., Mulhern, M. G., and Rootman, D. (2001). Tyrosinemia type II: nine cases of ocular signs and symptoms. Am J Ophthalmol 132, 522–527.CrossRefGoogle Scholar
  35. Maydan, G., Andresen, B.S., Madsen, P.P., Zeigler, M., Raas-Rothschild, A., Zlotogorski, A., Gutman, A., and Korman, S.H. (2006). TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping. J Inherit Metab Dis 29, 620–626.CrossRefGoogle Scholar
  36. Mehta, P.K., Hale, T.I., and Christen, P. (1993). Aminotransferases: demonstration of homology and division into evolutionary subgroups. Eur J Biochem 214, 549–561.CrossRefGoogle Scholar
  37. Meissner, T., Betz, R.C., Pasternack, S.M., Eigelshoven, S., Ruzicka, T., Kruse, R., Laitenberger, G., and Mayatepek, E. (2008). Richner-Hanhart syndrome detected by expanded newborn screening. Pediatr Dermatol 25, 378–380.CrossRefGoogle Scholar
  38. Minami-Hori, M., Ishida-Yamamoto, A., Katoh, N., Takahashi, H., and Iizuka, H. (2006). Richner-Hanhart syndrome: report of a case with a novel mutation of tyrosine aminotransferase. J Dermatol Sci 41, 82–84.CrossRefGoogle Scholar
  39. Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., and Olson, A.J. (1998). Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19, 1639–1662.CrossRefGoogle Scholar
  40. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–255.CrossRefGoogle Scholar
  41. Natt, E., Kida, K., Odievre, M., Di Rocco, M., and Scherer, G. (1992). Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II. Proc Natl Acad Sci U S A 89, 9297–9301.CrossRefGoogle Scholar
  42. Natt, E., Westphal, E.M., Toth-Fejel, S.E., Magenis, R.E., Buist, N.R., Rettenmeier, R., and Scherer, G. (1987). Inherited and de novo deletion of the tyrosine aminotransferase gene locus at 16q22.1-q22.3 in a patient with tyrosinemia type II. Hum Genet 77, 352–358.CrossRefGoogle Scholar
  43. Nose, S. (1984). A unified formulation of the constant-temperature molecular-dynamics methods. J Chem Phys 81, 511–519.CrossRefGoogle Scholar
  44. Nose, S., and Klein, M.L. (1983). Constant pressure molecular dynamics for molecular systems. Mol Phys 50, 1055–1076.CrossRefGoogle Scholar
  45. Nowicki, C., Hunter, G.R., Montemartini-Kalisz, M., Blankenfeldt, W., Hecht, H., and Kalisz, H.M. (2001). Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme. Biochim Biophys Acta 1546, 268–281.Google Scholar
  46. Oostenbrink, C., Villa, A., Mark, A.E., and van Gunsteren, W.F. (2004). A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem 25, 1656–1676.CrossRefGoogle Scholar
  47. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307–326.CrossRefGoogle Scholar
  48. Parrinello, M., and Rahman, A. (1981). Polymorphic transitions in single crystals: A new molecular dynamics method. J Appl Phys 52, 7182–7190.CrossRefGoogle Scholar
  49. Pasternack, S.M., Betz, R.C., Brandrup, F., Gade, E.F., Clemmensen, O., Lund, A.M., Christensen, E., and Bygum, A. (2009). Identification of two new mutations in the TAT gene in a Danish family with tyrosinaemia type II. Br J Dermatol 160, 704–706.CrossRefGoogle Scholar
  50. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612.CrossRefGoogle Scholar
  51. Rego, J.V., Murta, S.M., Nirdé, P., Nogueira, F.B., de Andrade, H.M., and Romanha, A.J. (2008). Trypanosoma cruzi: characterisation of the gene encoding tyrosine aminotransferase in benznidazoleresistant and susceptible populations. Exp Parasitol 118, 111–117.CrossRefGoogle Scholar
  52. Rossi, F., Han, Q., Li, J., Li, J., and Rizzi, M. (2004). Crystal structure of human kynurenine aminotransferase I. J Biol Chem 279, 50214–50220.CrossRefGoogle Scholar
  53. Schneider, G., Käck, H., and Lindqvist, Y. (2000). The manifold of vitamin B6 dependent enzymes. Structure 8, R1–R6.CrossRefGoogle Scholar
  54. Sivaraman, S., and Kirsch, J.F. (2006). The narrow substrate specificity of human tyrosine aminotransferase—the enzyme deficient in tyrosinemia type II. FEBS J 273, 1920–1929.CrossRefGoogle Scholar
  55. Sobrado, V.R., Montemartini-Kalisz, M., Kalisz, H.M., De La Fuente, M.C., Hecht, H.J., and Nowicki, C. (2003). Involvement of conserved asparagine and arginine residues from the N-terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases. Protein Sci 12, 1039–1050.CrossRefGoogle Scholar
  56. Touboul, T., Hannan, N.R., Corbineau, S., Martinez, A., Martinet, C., Branchereau, S., Mainot, S., Strick-Marchand, H., Pedersen, R., Di Santo, J., et al. (2010). Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 51, 1754–1765.CrossRefGoogle Scholar
  57. Vagin, A., and Teplyakov, A. (1997). MOLREP: an automated program for molecular replacement. J Appl Cryst 30, 1022–1025.CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Prajwalini Mehere
    • 1
  • Qian Han
    • 1
  • Justin A. Lemkul
    • 1
  • Christopher J. Vavricka
    • 1
    • 2
  • Howard Robinson
    • 3
  • David R. Bevan
    • 1
  • Jianyong Li
    • 1
    Email author
  1. 1.Department of BiochemistryVirginia TechBlacksburgUSA
  2. 2.CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  3. 3.Biology DepartmentBrookhaven National LaboratoryUptonUSA

Personalised recommendations