Incorporation of Two Oxygens from ¹⁸O₂ in the Epoxyquinone from the Dihydroxyacetanilide Epoxidase Reaction:  Evidence for a Dioxygenase Mechanism

Abstract

Dihydroxyacetanilide, 4, is oxidized to the 5R,6S-epoxyquinone, 3, by dihydroxyacetanilide epoxidase-I (DHAE I) from Streptomyces LL-C10037, without the assistance of an organic cofactor. 13C NMR analysis revealed that in the presence of 18O2 a full equivalent of 18O is incorporated at the epoxide. However, control reactions revealed the rapid exchange of the C-4 carbonyl with H2 18O. By coupling the DHAE I reaction with 2-acetamido-5,6-epoxy-1,4-benzoquinone oxidoreductase (AEBQOR I) from the same organism, NADP, and an NADPH regeneration system based on glucose 6-phosphate dehydrogenase, the epoxyquinol LL-C10037α, 1, was produced with ∼20% incorporation of a second 18O atom at the C-4 alcohol. Therefore, DHAE I is a dioxygenase with an epoxidation mechanism essentially the same as has been observed for the dihydrovitamin K epoxidation occurring during the mammalian vitamin K-dependent glutamate carboxylase reaction.

Aqueous Phosphoester Bond Cleavage of Dimethyl Phosphate by Cp2MoCl2: First Reported Case of Hydrolytic Cleavage on an Unactivated Phosphate Diester by an Organometallic Complex

“…This suggests no oxygen from the solvent was incorporated into the methanol which would give an 18 O isotope-induced 13 C shift of 0.04 ppm. 16 One mechanism consistent with the 31 P and 13 C NMR spectra at pD 4.0 is for a phosphoester bond cleavage pathway that proceeds mainly through P-O cleavage as opposed to C-O cleavage. An intermolecular attack on the CH 3 of DMeP would result in 50% 18 O incorporation in the free methanol at 49 ppm which was not seen in the 13 C NMR spectrum.…”

Enzymatic Chemistry of Cyclopropane, Epoxide, and Aziridine Biosynthesis

“…The former is an antitumor agent produced by Streptomyces LL-C10037, and the latter is an anticancer compound isolated from Streptomyces MPP 3051. 196–198 Studies on DHAE I have shown that this enzyme converts dihydroxyacetanilide ( 145 ) to the ( 5R , 6S )-epoxyquinone ( 147 ), with quantitative incorporation of 18 O (derived from 18 O 2 ) into the epoxide. 198 Control experiments demonstrated that the oxygen atom at C4 exchanges quickly with solvent.…”

The Biosynthesis of Epoxides

“…Unlike any of the epoxidases discussed so far, DHAE does not require the addition of any cofactors other than molecular oxygen for its activity to be reconstituted in vitro [78]. In contrast to cytochrome P450 enzymes and squalene epoxidase, DHAE is a dioxygenase, as it transfers both atoms of molecular oxygen to the substrate [80]. No reducing system is required for enzyme turnover as all electrons needed for the reduction of O 2 are directly derived from the substrate.…”

“…No reducing system is required for enzyme turnover as all electrons needed for the reduction of O 2 are directly derived from the substrate. In order to demonstrate the incorporation of both oxygen atoms into the product, the reaction has to be coupled to the next step in the biosynthetic pathway -the reduction of the carbonyl to the hydroxy group [80]. This procedure is necessary because of the fast exchange of the C4 oxygen with solvent water that would wash out any incorporated 18 O label [81].…”

“…This procedure is necessary because of the fast exchange of the C4 oxygen with solvent water that would wash out any incorporated 18 O label [81]. A mechanism involving a dioxetane intermediate has been proposed for the DHAE-catalyzed epoxidation of dihydroxyacetanilide that is based on the incorporation of 18 O into the epoxide as well as into the hydroxy group (Scheme 10.21) [80].…”