Study On Structure Characterisitics And Function Of Tyrosine Hydroxylase In Bombyx Mori And Tenebrio Molitor

Melanization is an oxidation-reduction reaction from phenolics to quinones andpigment. At first, tyrosine is hydroxylated and transformed into Dopa, and then, with theexistence of Polyphenol Oxidase and many other enzymes, Dopa was changed intomelanin. Both Tyrosine hydroxylase (TH) and Polyphenol Oxidase (PO) could inducethe production of dopa from tyrosine. TH is an aromatic amino acid hydroxylase, whichcould catalysis the formation of tyrosine by using tyrosine as its substrate andcombining with tetrahydrobiopterin. TH wildly exists in insets, mammals and humanbeings as an essential enzyme for metabolism. Many researchers have proved that POcould catalysis dopa to tyrosine as tyrosinase, At the same time, it participates thecatalytic oxidation of polyphenols with dual function. Some evidence showed that POplayed a very important role in the surface hardening of insets, production of pigments,wound cicatrization and resistance of pathogens. Some researchers focused too much onthe phenol oxidase in the past. According to the critical role in physiological functionsfor both PO and TH, therefore it will be very meaningful to analysis structuralproperties of TH.Here we take Tenebrio molitor and Bombyx mori for experimental materials. Firstwe analysis the major open reading frames sequence of TmTH (gene of PO fromTenebrio molitor) and BmTH (gene of TH from Bombyx mori), then bioinformaticsanalysis was performed. Afterwards we constructed the expression vectors from E. coliBL21and let them express the recombinant proteins. And finally, we got the targetproteins via affinity chromatography. The PO was separated and purified in thehemolymph of Tenebrio molitor, and the enzyme activity and its dynamic parameterswere detected in recombinant of TmTH in vitro, TmTH and PO in vivo respectively.Then the catalyze efficiency of both TmTH and PO was compared and analyzed.Besides, the impact of TmTH on the pigments production and hardening, lymphocytes melanization and its abnormalities in developments were observed by using theinhibitor of TH. Then the fluorescence spectra, activity and kinetic constants of theexpression protein are detected after construction mutant of BmTH, and purification ofits recombinant protein.The results showed that both the cloned TmTH and the BmTH by menns of thebioinformatics prediction belong to the tyrosine hydroxylase type II of Drosophilamelanogaster, with N-terminus containing the regulatory region of a period rich inacidic amino acids and the C-terminus containing a conserved hydroxylase catalyticdomain of aromatic family of amino acid.There is no significant difference about the kinetic parameters between therecombinant TmTH and the TmTH in its body, so the recombinant TmTH can be usedto replace natural TmTH for related research. TmTH possess lower affinity of tyrosineand much lower catalytic efficiency compared with PO. The decrease of the TmTHactivity could lead the black level of the hemolymph to decline, the pigmentation toweaken in cultured epidermal tissue, and finally caused the malformed development andmetamorphosis of Tenebrio molitor. The AA419of BmTH catalytic amino acids wasmutated from aspartate to glycine mutants and the N-terminal regulatory region wasdeleted, which made the activity decrease, the three-dimensional structural change.Although the tyrosine affinity of the restructured BmTH was not significant comparedwith PO, but the catalytic efficiency is lower than that of PO. In summary, the structuralproperties of the TH in insects are similar to mammals, playing an important role in theepidermal pigment formation, development, metamorphosis and immune defense ininsects. The results of the study can enrich the epidermal tanning theory and immunethe theory of insects, and may lay the foundation for the function and mechanismregulation of TH in further study.