Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism

Abstract

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4 reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs that catalyse 1,2-aldehyde and 1,2-iminium reductions suggest how the canonical ADH active site can be modified to carry out atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Recycling upstream redox enzymes expands the regioselectivity of cycloaddition in pseudo-aspidosperma alkaloid biosynthesis

“…We isolated isotopically labeled (–)-tabersonine ( 2 ), Ψ-tabersonine ( 5 ), and Ψ-vincadifformine ( 9 ) and showed that the deuterium labels were incorporated at carbon 19 for (–)-tabersonine ( 2 ) as expected, 13 carbons 19 and 21 for Ψ-tabersonine ( 5 ), carbons and 19, 21, 15 for Ψ-vincadifformine ( 9 ) ( Figure 4) . We performed CD spectroscopy of these isolated products and assigned the stereochemistry as (+)-Ψ-tabersonine ( 5 ) and (+)-Ψ-vincadifformine ( 9 ), which is the expected stereochemistry based on the downstream ibophyllidine products (Figure S16).…”

“…The (-)-tabersonine (2) product produced by TiTabS had no deuterium incorporation, as expected; for-mation of Ψ-tabersonine (5) and Ψ-vincadifformine (9) from angryline (1a) showed incorporation of one and deuterium atoms respectively and was strictly dependent upon the presence of both reductase, TiDPAS1, and dase, PAS (Figure S15). We isolated isotopically labeled (-)-tabersonine (2), Ψ-tabersonine (5), and Ψ-vincadifformine (9) and show that the deuterium labels were incorporated at carbon 19 for (-)-tabersonine (2) as expected, 13 carbons 19 and for Ψ-tabersonine (5), carbons and 19, 21, 15 for Ψ-vincadifformine (9) (Figure 4). We performed CD spect copy of these isolated products and assigned the stereochemistry as (+)-Ψ-tabersonine ( 5) and (+) vincadifformine (9), which is the expected stereochemistry based on the downstream ibophyllidine products (F ure S16).…”

“…With TiTabS, we saw formation of the (-)tabersonine (2) product with a mass consistent with incorporation of one deuterium, as expected from the action of DPAS acting on precondylocarpine acetate (7) (Figure 4). 13 However, when TiCorS was substituted for TiTabS, the resulting Ψ-tabersonine (5) product had a mass consistent with incorporation of two deuterium atoms (Figure 4 and Figure S14), clearly demonstrating that formation of Ψ-tabersonine (5) from stemmadenine acetate (6) requires two reduction steps. Furthermore, the Ψ-vincadifformine (9) product showed a mass incorporation of three deuterium atoms (Figure 4 and Figure S14).…”

“…Stemmadenine acetate ( 6) is first oxidized by flavin dependent enzyme precondylocarpine acetate synthase (PAS) 5 to generate precondylocarpine acetate and then undergoes a 1,4-iminium reduction by the medium chain alcohol dehydrogen dihydroprecondylocarpine acetate synthase (DPAS) (Figure 1). 13 The resulting reduced unstable prod dihydroprecondylocarpine acetate (8), undergoes elimination of an acetoxy group to yield dehydrosecodine which is then captured by one of the cyclases (Figure 1).…”

“…Therefore, we performed the reaction in the presence of isotopically labelled NADPH (pro-(R)-NADPD) to deter-mine where the deuteride is incorporated in Ψ-tabersonine (5). 13,17,18 We incubated stemmadenine acetate (6) with CrPAS, TiDPAS1, and TiCorS or TiTabS, along with pro-(R)-NADPD, which is required for DPAS reduction (Figure S14). With TiTabS, we saw formation of the (-)tabersonine (2) product with a mass consistent with incorporation of one deuterium, as expected from the action of DPAS acting on precondylocarpine acetate (7) (Figure 4).…”