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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Molecular Modeling and Site-Directed Mutagenesis Reveal the Benzylisoquinoline Binding Site of the Short-Chain Dehydrogenase/Reductase Salutaridine Reductase^1,[W],[OA]

Leibniz-Institute of Plant Biochemistry, D–06120 Halle, Germany

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 /-folding pattern of SDRs, including the four additional helices F'-1 to F'-4 assumed to prevent the dimerization of the monomeric short-chain dehyrogenases/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 E' 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.

² Present address: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jörg Ziegler (joerg.ziegler{at}ucalgary.ca).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.095166

^* Corresponding author; e-mail joerg.ziegler{at}ucalgary.ca; fax 1–403–289–9311.

Received December 20, 2006; accepted February 18, 2007; published March 2, 2007.