Preparation of regulatory domain of tyrosine hydroxylase for NMR studies


M. Kopecka 1,2, L. Rezabkova 1,2, Z. Tosner 1, M. Stepanek 1, V. Obsilova 2, T. Obsil 1,2

 

1 Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic

2 Institute of Physiology, Academy of Science of Czech Republic, 14220 Prague, Czech Rep.

kopeckamirka@tiscali.cz

 

Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids, class oxydoreductases and subgroup oxygenases. This enzyme catalyses key step in the biosynthesis of catecholamine neurotransmitters the conversion of the tyrosine to the 3,4-dihydroxyfenylalanine. We can found it mainly in cells of the adrenal gland, in the heart, in the liver, in gonads and in the central nervous system [1, 2].

The tyrosine hydroxylase has the homotetrameric structure and contains three diverse structural domains: N-terminal regulatory domain, catalytic domain and C-terminal tetramerization domain [3]. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. All phosphorylation sites (Ser-8, Ser-19, Ser-31 and Ser‑40) are located within the regulatory domain [2]. Phosphorylation at Ser-40 by cyclic AMP-dependent protein kinase (PKA) induces the most potent activation of tyrosine hydroxylase. It has been proposed that phosphorylation of Ser-40 alters the conformation of the regulatory domain and its interaction with the catalytic domain. Phosphorylation at Ser‑19 induces binding of the 14‑3‑3 protein, which affects the structure of the regulatory domain and protects it against dephosphorylation (at phosphorylated Ser-40) and its degradation [4, 5].

Since the structure of the regulatory domain is still unknown we decided to perform its structural characterization using NMR techniques. The regulatory domain of tyrosine hydroxylase was expressed as six-His-tag fusion protein by IPTG induction for 12 h at 20 C and purified from Escherichia coli BL21(DE3). Its purification consists of two steps: the chelating chromatography and the size-exclusion chromatography on Superdex 200 column. The dynamic light scattering and the 1H nuclear magnetic resonance were used to verify that the recombinant protein is not aggregated and can be used for further experiments.

 

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2. P. F. Fitzpatrick, Annu Rev Biochem 68, (1999), 355381.

3. S.C. Daubner, D.L. Lohse, P.F. Fitzpatrick, Protein Sci 2, (1993), 14521460.

4. C. Itagaki, T. Isobe, M. Taoka, T. Natsume, N. Nomura, T. Horigome, S. Omata, H. Ichinose, T. Nagatsu, L.A. Greene, T. Ichimura, Biochemistry 38, (1999), 1567315680.

5. V. Obsilova, E. Nedbalkova, J. Silhan, E. Boura, P. Herman, J. Vecer, M. Sulc, J. Teisinger, F. Dyda, T. Obsil, Biochemistry 47, (2008), 1168-1777.

 

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Grant 28510 of the Grant Agency of the Charles University, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.