Further Insights on the <i>Datura innoxia</i> Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Abstract

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6 β -hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6 β -hydroxylase (H6H), a 2-o xoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum , preventing the accumulation of anisodamin e and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel , we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein . We also address the enantiomeric preference of DiH6H based on molecular modeling.