This review provides a renewed look at the genus Digitalis. Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used (“Digitalis” and “Foxglove” were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here. Intriguing new results emerged from studies related to the phylogeny and taxonomy of the genus as well as to the biosynthesis and potential medicinal uses of the key active compounds, the cardiac glycosides. Several Eastern and Western Foxgloves were studied with respect to their propagation in vitro. In this context, molecular biology tools were applied and phytochemical analyses were conducted. Structure elucidation and analytical methods, which have experienced less exciting progress, will not be considered here in great detail. ^* Dedicated to Professor Dr. Max Wichtl in recognition of his outstanding contribution to pharmacognosy research.

The amazing variability of plant metabolism and its rapid divergence during evolution pose fundamental questions as to the driving forces, mechanisms, and players in metabolic differentiation. This review examines concepts that help us understand adaptive pathway evolution, with a particular emphasis on plant specialized metabolism, previously often termed secondary metabolism. Following a general introduction to pathway and metabolite evolution, the focus is directed to enzyme promiscuity and its classification. Promiscuous enzymes (or substrates), 'silent' elements of the metabolome, and the 'underground metabolism' may be used and combined to evolve 'new' metabolic pathways. It appears that new pathways rarely appear from scratch, but instead emerge from 'floppy' enzymes and elements of a 'messy' metabolism, and in this way a range of metabolites is generated, some of which may provide benefits to the plant.

Vein Patterning 1 (VEP1)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase superfamily of proteins. They are characterized by a set of highly conserved amino acids in the substrate-binding pocket. All PRISEs are capable of reducing the activated C=C double bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis. Here, recombinant forms of PRISEs of Arabidopsis thaliana and Digitalis lanata were modified using site-directed mutagenesis (SDM). In rDlP5βR, a set of highly conserved amino acids in the vicinity of the catalytic center was individually substituted for alanine resulting in considerable to complete loss of enone reductase activity. F153 and F343, which can be found in most PRISEs known, are located at the outer rim of the catalytic cavity and seem to be involved in substrate binding and their role was addressed in a series of SDM experiments. The wild-type PRISE accepted progesterone (large hydrophobic 1,4-enone) as well as 2-cyclohexen-1-one (small hydrophilic 1,4-enone), whereas the double mutant rAtP5βR_F153A_F343A converted progesterone much better than the wild-type enzyme but almost lost its capability of reducing 2-cyclohexen-1-one. Recombinant Draba aizoides P5βR (rDaP5βR) has a second pair of phenylalanines at position 156 and 345 at the rim of the binding site. These two phenylalanines were introduced into rAtP5βR_F153A_F343A and the resulting quadruple mutant rAtP5βR_F153A_F343A_V156F_V345F partly recovered the ability to reduce 2-cyclohexen-1-one. These results can best be explained by assuming a trapping mechanism in which phenylalanines at the rim of the substrate-binding pocket are involved. The dynamic behavior of individual P5βRs and mutants thereof was investigated by molecular dynamics simulations and all calculations supported the ‘gatekeeper’ role of phenylalanines at the periphery of the substrate-binding pocket. Our findings provide structural and mechanistic explanations for the different substrate preferences seen among the natural PRISEs and help to explain the large differences in catalytic efficiency found for different types of 1,4-enones.

Small or specialized natural products (SNAPs) produced by plants vary greatly in structure and function, leading to selective advantages during evolution. With a limited number of genes available, a high promiscuity of the enzymes involved allows the generation of a broad range of SNAPs in complex metabolic networks. Comparative metabolic studies may help to understand why—or why not—certain SNAPs are produced in plants. Here, we used the wound-induced, vein patterning regulating VEP1 (AtStR1, At4g24220) and its paralogue gene on locus At5g58750 (AtStR2) from Arabidopsis to study this issue. The enzymes encoded by VEP1-like genes were clustered under the term PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) as it was previously demonstrated that they are involved in cardenolide and/or iridoid biosynthesis in other plants. In order to further understand the general role of PRISEs and to detect additional more “accidental” roles we herein characterized A. thaliana steroid reductase 1 (AtStR1) and compared it to A. thaliana steroid reductase 2 (AtStR2). We used A. thaliana Col-0 wildtype plants as well as VEP1 knockout mutants and VEP1 knockout mutants overexpressing either AtStR1 or AtStR2 to investigate the effects on vein patterning and on the stress response after treatment with methyl vinyl ketone (MVK). Our results added evidence to the assumption that AtStR1 and AtStR2, as well as PRISEs in general, play specific roles in stress and defense situations and may be responsible for sudden metabolic shifts.