Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in the present work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9, 10-dihydroxystearate, that we identified via its methyl ester through gas chromatography/mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 18O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9, 10-dihydroxystearate, which presents a likely explanation for its accumulation to approximately 0.7% in castor oil, of which the biosynthetic origin had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of diiron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.