Publications

• Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase.

  Geissler R., Brandt W., Ziegler J.

  Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of hum ... >> More

  Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical alpha/beta-folding pattern of SDRs, including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization of the monomeric short-chain dehydrogenases/reductases. Site-directed mutagenesis of asparagine-152, serine-180, tyrosine-236, and lysine-240 resulted in enzyme variants with strongly reduced performance or inactive enzymes, showing the involvement of these residues in the proton transfer system for the reduction of salutaridine. The strong preference for NADPH over NADH could be abolished by replacement of arginine residues 44 and 48 by glutamic acid, confirming the interaction between the arginines and the 2'-phosphate group. Docking of salutaridine into the active site revealed nine amino acids presumably responsible for the high substrate specificity of salutaridine reductase. Some of these residues are arranged in the right position by an additional alphaE' helix, which is not present in SDRs analyzed so far. Enzyme kinetic data from mutagenic replacement emphasize the critical role of these residues in salutaridine binding and provide the first data on the molecular interaction of benzylisoquinoline alkaloids with enzymes. << Less

  Plant Physiol. 143:1493-1503(2007) [PubMed] [EuropePMC]

• Removal of substrate inhibition and increase in maximal velocity in the short chain dehydrogenase/reductase salutaridine reductase involved in morphine biosynthesis.

  Ziegler J., Brandt W., Geissler R., Facchini P.J.

  Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length ... >> More

  Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel alpha-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR. << Less

  J. Biol. Chem. 284:26758-26767(2009) [PubMed] [EuropePMC]

• Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis.

  Ziegler J., Voigtlander S., Schmidt J., Kramell R., Miersch O., Ammer C., Gesell A., Kutchan T.M.

  Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the an ... >> More

  Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism. << Less

  Plant J. 48:177-192(2006) [PubMed] [EuropePMC]