Studies On The Expression, Purification, Structure And Properties Of Tyrosine Hydroxylase From Bombyx Mori

The tyrosine hydroxylase could catalyze L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), DOPA is the precursors of some biologicallyactive compounds, including dopamine, melanin, adrenaline and other substances. TH isthe rate-limiting enzyme in the synthesis of Catecholamine derivatives, enzymaticreaction of which need the existence of tetrahydrobiopterin as the coenzyme and Fe2+for agonist. It is widely present in mammals, humans and insects. Some studies havefound that catecholamines play a critical important role in the coloring of the insectepidermis tanning, wound healing, the exoskeleton hardened and immune black.Furthermore, as the key enzyme of the catalytic synthesis of catecholamines, TH isessential enzymes in insect metabolism. Therefore, TH must have an importantphysiological function in insects.At present, some reports about tyrosine hydroxylase in insect have been published,especially on Drosophila melanogaster. However, Bombyx mori is only one kind ofLepidoptera insect with complete genome sequencing and its economic benifits. Thisstudy will take the Bombyx mori as basic experimental materials, and preliminaryresearche will be conducted on the tissue location, structure and properties of thetyrosine hydroxylase in Bombyx mori.First, the cDNA sequence of the Bombyx mori tyrosine hydroxylase (BmTH) wasgot and then sequence analyzing showed that N-terminal of BmTH’s had the AA1–AA150as the regulatory region, Contains74amino acid fragments, the fragments richin acidic amino acids. Also, it contains the C-terminal region with a conserved aromaticamino acid and the binding sites of tetrahydrobiopterin and Fe2+which is as the catalyticregion by hydroxylase superfamily structure (AA224-AA521). BmTH was transferredinto the BL21, after being connected into the prokaryotic expression vector PET-28(+).By optimizing expression conditions, we found that the BmTH prokaryotic expressionwould reach the highest level with the optimized conditions:1mM IPTG existence and 18℃for14h. BmTH, as a soluble form, could be isolated and purified by affinitychromatography (MCAC) and was active recombinant protein with high-purity. Themolecular weight of BmTH is about64KD, examined by SDS-PAGE. The expressionlevels in the head, body, silk gland and gonads were detected by semi-quantitative PCR.The results showed that BmTH could express in all of the above organs. By immunefluorescence localization, we found BmTH expressed in the cytoplasm of BmN cells.Most of BmTH located in the fat body near the endepidermis and endocuticle.Meanwhile, we established a high-performance liquid UV method to detect the activityof tyrosine hydroxylase in Bombyx mori. This method can be used to measure tyrosinehydroxylase (TH) activity of insect fast, stably and much sensitively. Furthermore, wesuccessfully got the two mutants BmTH-D419G and BmTH-A-of the BmTH by usingoverlapping PCR technique and we also found that the BmTH activity decreased whilethe Asp419mutated into Gly, and intrinsic fluorescence spectra of some of the blue shiftwas found and fluorescence intensity was decreased, the D419G mutation led to BmTHstructural change, and affected the activity of the enzyme, the lack of the N-terminus ofthe regulatory regions reduced the activity of the enzyme in a large degree, and intrinsicfluorescence spectra of some of the blue shift was found and fluorescence intensity wasdecreased,thus the lack of potential regulatory regions could affect the stability of thetetramer structure.Generally speaking, this study laid the basic foundation for the further study on thestructure, function and regulation of the tyrosine hydroxylase in Bombyx mori.