Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Abstract

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 ␣-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.

Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

“…It should also be noted that the BetB mutant proteins with reduced or eliminated substrate inhibition showed a significant decrease in k cat /K m . Similar effects were also demonstrated in substrate inhibition studies with other enzymes (4,13), and in some cases they might be associated with the reduced substrate affinity of mutant proteins.…”

“…The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites. Recent biochemical studies have demonstrated that substrate inhibition of several dehydrogenases can be significantly reduced or eliminated by single mutations in the enzyme active site, which can be accompanied by a reduction or increase in the reaction rate and usually correlates with a reduction of substrate affinity (increasing K m ) (4,7,10,13). For example, the mutation S163L in human lactate dehydrogenase eliminates substrate inhibition with a minor effect on its turnover rate, whereas in Bacillus stearothermophilus lactate dehydrogenase the D38R mutation reduces substrate inhibition 3-fold (4,14).…”

“…For dehydrogenases, several molecular mechanisms of substrate inhibition have been proposed, including the formation of a covalent adduct between the oxidized forms of substrate and cofactor, allosteric inhibition (which occurs away from the active site, e.g., in D-3-phosphoglycerol dehydrogenase from Mycobacterium tuberculosis), and the formation of a nonproductive enzyme complex with cofactor and/or substrate (6)(7)(8)(9)(10)(11)(12)(13). The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites.…”

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

“…Location and Effects of SalR Mutations-In a previous study (30), a homology modeling and mutagenesis study was performed on SalR. From this analysis, it was suggested that the apparent substrate inhibition observed at high salutaridine concentrations is due to a second, nonproductive, and overlapping binding mode in the active site.…”

Crystal Structure of a Lipoxygenase in Complex with Substrate

“…Substrate inhibition has been studied in other enzymes (20,(35)(36)(37), and its analysis can be complex (32,38,39). We observed that the R182A data are best explained by cooperative substrate inhibition (Equation 1).…”