Removal of substrate inhibition and increase in maximal velocity in the short chain dehydrogenase/reductase salutaridine reductase involved in morphine biosynthesis.
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.
Ziegler
Jörg
J
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Brandt
Wolfgang
W
Geissler
René
R
Facchini
Peter J
PJ
D053898:Biosynthetic Pathways; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009019:Morphinans / Q000378:metabolism / Q000494:pharmacology; D009020:Morphine / Q000096:biosynthesis* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D009019:Morphinans; D010940:Plant Proteins; D009249:NADP; D009020:Morphine; C009270:salutaridine; D010088:Oxidoreductases; C520720:salutaridine reductase, Papaver