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 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate 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 anisodamine 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.

Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna

“…Two remaining metabolites, T20 and T21, lack putative annotations (Table S8 ). Although the exact mass of T20 [M+H] + , m/z 306.1699 corresponds to C 17 H 24 NO 4 + , consistent with hydroxyhyoscyamine and hydroxylittorine, the fragment ions m/z 200.13 and 154.12 are inconsistent with this annotation as these metabolites yield m/z 158.12 and m/z 140.12 fragment ions (Schlesinger et al ., 2021 ).…”

“…Two remaining metabolites, T20 and T21, lack putative annotations (Table S8). Although the exact mass of T20 [M+H] + , m/z 306.1699 corresponds to C 17 H 24 NO 4 + , consistent with hydroxyhyoscyamine and hydroxylittorine, the fragment ions m/z 200.13 and 154.12 are inconsistent with this annotation as these metabolites yield m/z 158.12 and m/z 140.12 fragment ions (Schlesinger et al, 2021). Tandem mass spectrometry of ions increased in AbPyKS-silenced plants revealed that 36 metabolites gave rise to an m/z 84.08 fragment ion (C 5 H 10 N + ) consistent with metabolites that contain a methylpyrrolidine moiety (Fig.…”