Purification and characterization of dopamine-secologanin condensing enzyme from alangium salvifolium wang ssp. hexapetalum wang leaves

Abstract

Dopamine-secologanin condensing enzyme, the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified from the leaves of Alangium salviifolium Wang. The enzyme was purified to apparent electrophoretic homogeneity by ammonium sulfate precipitation, ultrafiltration and three subsequent column chromatography steps. The isolated enzyme is a single polypeptide with M, 30,000 and has a pH optimum at 7.5 and a temperature optimum at 37°c. The apparent Km value for dopamine and secologanin are 0.69 mM and 0.92 mM, respectively. The Vmax for dopamine and secologanin are 7.09 and 8.33 pkat/mg protein, respectively. The enzyme has high substrate specificity to dopamine; neither tyramine nor tryptamine are utilized by the enzyme. No substrate inhibition was observed. The enzyme activity is inhibited by alangimarckine and dehydroalangimarckine with similar IC50 value approximately of 10 mM. The enzymatic product was confirmed to be demethylalangiside which is the spontanous lactamization product of (R)-deacetylipecoside. From these results, the dopamine-secologanin condensing enzyme was named “deacetylipecoside synthase” which presumeably catalyzes the provision of (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that possess also (R)-configuration at the same chiral center.