Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

Abstract

The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of ϳ1.9 Å in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large "flap"-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.The narcotic analgesic morphine and the antitussive codeine are the most important active alkaloids from the opium poppy (Papaver somniferum L.). The tetracyclic morphinan salutaridine is an intermediate in morphine and codeine biosynthesis. P. somniferum salutaridine reductase (SalR 2 ; EC 1.1.1.248) reduces the C-7 keto group of salutaridine to the C-7 S-configuration hydroxyl group of salutaridinol using the 4-pro-S-hydride of NADPH. Only the S-configuration of salutaridinol is biologically active, as demonstrated by its transformation to morphine and codeine in vivo (1, 2). SalR was purified from a plant cell culture and characterized by Gerardy and Zenk (3). The SalR cDNA was identified during a crossspecies comparison of gene expression profiles between 16 different Papaver species (4).SalR belongs to the short chain dehydrogenase/reductase (SDR) family. The SDR family of proteins has a single domain composed of a parallel ␣/␤-fold with a Rossmann fold motif for NAD(P)(H) binding. A central twisted parallel ␤-sheet is flanked by three ␣-helices on each side (5). Among the known structures of proteins belonging to the SDR family, human CBR1 (carbonyl reductase 1) has the highest sequence identity to SalR (105 of 311 amino acid residues) (6, 7) and is a drug-metabolizing enzyme. Human carbonyl reductases react with a number of organic compounds such as steroids, prostaglandins, xenobiotics, and S-nitrosoglutathione (8 -10). In contrast, salutaridine is the only known substrate of plantderived SalR (3, 4). SalR has a 36-residue insertion (Ser 105 -Glu 140 ) that does not exist in animal carbonyl r...

Discovery of a Short‐Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid

“…To understand the mechanism of VAS better, the crystal structure was solved (Table and Figure ; PDB ID: 5O98). It closely resembles the structure of salutaridine reductase from Papaver somniferum (SalR; PDB ID: 3O26; i.e., the molecular replacement template) and the recently determined structures of two SDRs from Mentha piperita : menthone‐neomenthol reductase (MNMR; e.g., PDB ID: 5L53) and isopiperitenone reductase (IPR; e.g., PDB ID: 5LCX; Figure S10). Crystallization of VAS was carried out with and without the oxidized cofactor NADP + .…”

“…The structure was solved by molecular replacement by using PHASER and the crystal structure of SalR (PDB ID: 3O26), with which it shares 53 % amino acid sequence identity, as template. The asymmetric unit contained two monomers, corresponding to an estimated solvent content of 44 %.…”

Fenhexamid Resistance in the Botrytis Species Complex, Responsible for Grey Mould Disease

“…2. Predicted 3D structure of the 3-ketoreductase (Erg27) of Botrytis cinerea based on the alignement with Salutaridine Reductase From Papaver Somniferum (Higashi et al, 2011). The cofactor (NADHP) binding site and the active sites are highlighted in cyan and purple respectively.…”

Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

“…Crystallographic methodology, data refinement statistics, and detailed structural descriptions can be found in the Supporting Information (Table S2 and associated discussion). The crystal structures of apo‐IPR and the 3 a ‐ and β‐cyclocitral‐bound complexes were solved by molecular replacement using the known SalR crystal structure (PDB 3O26; 1.2 and 1.7 Å resolution, respectively; Table S2) 10. The presence of clear density in the F 0 − F c map for NADP + (Figure 1 B) suggested IPR had scavenged it from host cells during protein expression.…”