Identification of clerodane diterpene modifying cytochrome P450 (CYP728D26) in Salvia divinorum - en route to psychotropic salvinorin A biosynthesis.

A newly identified enzyme, CYP728D26, catalyzes a specific oxygenation step in the biosynthesis of salvinorin A, a potent κ-opioid receptor agonist from the hallucinogenic plant Salvia divinorum. This enzyme acts on crotonolide G, a compound with a clerodane backbone, and has a Michaelis constant (Km) of 13.9 μM. A close relative, CYP728D25, shows activity only in yeast overexpression systems but not in purified assays, highlighting the need for cautious interpretation of such results. Understanding this pathway could enable biomanufacturing of semi-synthetic derivatives for developing alternative, non-opioid pain relievers targeting the κ-opioid receptor.

Study at a glance

Abstract

Salvia divinorum is a hallucinogenic plant native to the Oaxaca in Mexico. The active ingredient for psychotropic effects in this plant is salvinorin A, a potent and highly selective κ-opioid receptor agonist. Salvinorin A is distinct from other well-known opioids, such as morphine and codeine, in that it is a non-nitrogenous diterpenoid with no affinity for μ-opioid receptor, the prime receptor of alkaloidal opioids. A terpene opioid that selectively targets a new opioid receptor (κ-opioid receptor) can be instrumental in developing alternative analgesics. Elucidation of the salvinorin A biosynthetic pathway can help bio-manufacture diverse semi-synthetic derivatives of salvinorin A but, to date, only two enzymes in the Salvinorin A pathway have been identified. Here, we identify CYP728D26 that catalyzes a C18 oxygenation on crotonolide G, which bears a clerodane backbone. Biochemical identity of CYP728D26 was validated by in vivo reconstitution in yeast, 1H- and 13C-NMR analyses of the purified product, and kinetic analysis of CYP728D26 with a Km value of 13.9 μM. Beyond the single oxygenation on C18, collision-induced dissociation analysis suggested two additional oxygenations are catalyzed by CYP728D26 to form crotonoldie G acid, although this carboxylic acid form is a minor product. Its close homologue CYP728D25 exhibited a C1-hydroxylation on the clerodane backbone in a reconstituted yeast system. However, CYP728D25 showed no activity in in vitro assays. This result implies that catalytic activities observed from overexpression systems should be interpreted cautiously. This work identified a new CYP catalyst and advanced our knowledge of salvinorin A biosynthesis.