The Expression And Functional Identification Of Anisodus Acutangulus Hyoscyamine 6 β-hydroxylase

Tropane alkaloids, including hyoscyamine, anisodine and scopolamine, arewidely found in Datura,Belladonna and other Solanaceae plants. As these alkaloidshave a strong anticholinergic activity and act in the parasympathetic nervous system,hyoscyamine and scopolamine have long been widely used. In the aspects of medicalvalue of these alkaloids, the scopolamine is about 10 times higher than hyoscyamine.However, scopolamine is much lower than hyoscyamine in Solanaceae plants.Therefore, it is important that production of high medical value medicine such asscopolamine meet the needs of requirements the clinical needs. Anisodus acutangulus,produced in the Yunnan people, has a long history and has been listed as endangeredspecies, the total alkaloid content compared to other Solanaceae plants can reachabove 1%.In the alkaloid metabolic pathway of solanaceae plants, scopolamine is catalyzedby hyoscyamine-6β-hydroxylase form hyoscyamine through anisodine as theintermidiate. The protein H6H belongsα-ketoglutarate-dependent dioxygenase family.It is a bifunctional enzyme, which can hydroxylation the C6 of hyoscyamine to formanisodamine, then epoxidized anisodine C-6 position and C-7 to form of scopolamine.The A.acutangulus cDNA sequene H6H has successfully obtained by thelaboratory. We construct H6H gene (cDNA) into the prokaryotic expression vectorpET30a and pGEX4T-1, so the AaH6H proteins is a label with His or GST tag.AaH6H can be expressed in E.coli by IPTG induction. Through the analysis ofaffinity chromatography purification and Western Blot, we confirmed that therecombinant AaH6H can be expressed as a soluble form. With the exression ofrecombinant protein, hyoscyamine have gradually been transformed into anisodamineand scopolamine. The results show that: AaH6H is a bifunctional enzyme, with bothhydroxylase activity (hyoscyamine into anisodine ) and epoxidation activity(anisodine into scopolamine). We study the function of AaH6H, this will help us product the high medical valuescopolamine in industrial production to meet clinical needs.