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Published on March 2, 2007; 10.1104/pp.106.095166

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Received December 20, 2006
Accepted February 18, 2007

Molecular Modelling and Site Directed Mutagenesis Reveals the Benzylisoquinoline Binding Site of the Short Chain Dehydrogenase / Reductase Salutaridine Reductase

René Geißler , Wolfgang Brandt , and Jörg Ziegler *

Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle / Germany

* Corresponding author; email: joerg.ziegler{at}ucalgary.ca.

Recently, the NADPH dependent short chain dehydrogenase / reductase salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum (Ziegler et al., 2006). In this report, a homology model of the Papaver bracteatum homologue was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical {alpha}/{beta} folding pattern of short chain dehydrogenases / reductases including the four additional helices {alpha}F`-1 to {alpha}F`-4 assumed to prevent the dimerization of the monomeric short chain dehyrogenases / reductases. Site directed mutagenesis of Asn-152, Ser-180, Tyr-236, and Lys-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 glutamate, 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 {alpha}E`-helix, which is not present in short chain dehydrogenases / reductases 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.




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