Enzymatic Epoxidation: II. COMPARISON BETWEEN THE EPOXIDATION AND HYDROXYLATION REACTIONS CATALYZED BY THE ω-HYDROXYLATION SYSTEM OF PSEUDOMONAS OLEOVORANS

Abstract

The ω-hydroxylation enzyme system of Pseudomonas oleovorans has been shown by others to catalyze the methyl group hydroxylation of alkanes and fatty acids. We have recently reported that this same enzyme system also catalyzes the epoxidation of alkenes. In the present study, a comparison of the hydroxylation and epoxidation reactions revealed that both require the three protein components (reductase, rubredoxin, and hydroxylase) of the ω-hydroxylation system. The substrate 1,7-octadiene is converted to both 7,8-epoxy-1-octene and 1,2–7,8-diepoxyoctane, whereas 1-octene is oxidized to both 7-octene-1-ol and 1,2-epoxyoctane. In addition, both 7,8-epoxy-1-octene and 1,2-epoxyoctane are oxidized by the enzyme system. Epoxidation, as found previously for hydroxylation, requires molecular oxygen, and NADPH does not substitute for NADH in either reaction. Both reactions are inhibited by cyanide and exhibit a similar pH dependence. A 1:1 molar stoichiometry was found between the amount of NADH oxidized and the amount of 7,8-epoxy-1-octene produced from 1,7-octadiene. In another experiment a 1:1 stoichiometry also was observed when the disappearance of the substrate 7,8-epoxy-1-octene was correlated with the amount of NADH oxidized. In the presence of excess O2, NADH, reductase, and rubredoxin, a competition was observed between octane and 1,7-octadiene for the hydroxylase component. Although it has not been possible so far to purify the hydroxylase to homogeneity, this study indicates that epoxidation and hydroxylation are mechanistically similar and thus may involve the same species of activated oxygen.