Purification and characterization of hyoscyamine 6β‐hydroxylase from root cultures of Hyoscyamus niger L.

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Abstract
Hyoscyanamine 6.beta.-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6.beta.-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. and Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average M4 of 41000 as determiend by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 .mu.M and 43 .mu.M. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate and was not replaced by any of ten other oxo acids tests, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 .mu.M and 90 .mu. M. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehytdrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 .mu.M). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6.beta.-hydroxylase.