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.

Pharmacologically important tropane alkaloids such as hyoscyamine, and scopolamine, are synthesized in the roots of several Solanaceae and also accumulate in their shoots. The tropane alkaloids content in roots of D. innoxia seedlings was almost 3-fold higher than in their shoots. Moreover, scopolamine accounted for 77 % of the total tropane alkaloid content in shoots as compared to only 20 % in roots. All candidate genes in the scopolamine biosynthetic pathway were significantly expressed in D. innoxia mature roots with limited expression in mature leaves. Using exogenous isotopically labeled 2H5 L-phenylalanine, we show here that root tissues, but not detached shoots, readily biosynthesize scopolamine. Tropane alkaloids newly synthesized by roots, and preferentially scopolamine, were further transported to shoots. Our evidence indicates that D. innoxia seedlings have the ability to synthesize tropane alkaloids in their roots and differentially transport them to the shoots.

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6β-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.

Volatile esters contribute to the aroma and flavor of many fruits but are normally absent in grape berries (Vitis vinifera L.). To examine the biosynthetic potential of grape berries to form volatile esters, berry sections were incubated with exogenous L-Phe, L-Leu or L-Met. In general, amino-acid incubation caused the accumulation of the respective aldehydes and alcohols. Moreover, L-Leu incubation resulted in the accumulation of 3-methylbutyl acetate and L-Phe incubation resulted in the accumulation 2-phenylethyl acetate in 'Muscat Hamburg' but not in the other grape accessions. Exogenous L-Met administration did not result in volatile esters accumulation but the accumulation of sulfur volatile compounds such as methional and dimethyl disulfide was prominent. Berry-derived cell-free extracts displayed differential alcohol acetyltransferase activities and supported the formation of 3-methylbutyl acetate and benzyl acetate. 2-Phenylethyl acetate was produced only in 'Muscat Hamburg' cell-free extracts. VvAAT2, a newly characterized gene, was preferentially expressed in 'Muscat Hamburg' berries and functionally expressed in E. coli. VvAAT2 possesses alcohol acetyltransferase activity utilizing benzyl alcohol, 2-phenylethanol, hexanol or 3-methylbutanol as substrates. Our study demonstrates that grape berries have a concealed potential to accumulate volatile esters and this process is limited by substrate availability.