Fjord-region Benzo[g]chrysene-11,12-dihydrodiol and Benzo[c]phenanthrene-3,4-dihydrodiol as Substrates for Rat Liver Dihydrodiol Dehydrogenase (AKR1C9): Structural Basis for Stereochemical Preference

This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k c a t/K m 100 times greater than that observed with the commonly studied bay-region benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[g]C-11,12-dihydrodiol, benzo[c]phenanthrene-3,4-dihydrodiol (B[c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[a]P-7,8-dihydrodiol and B[c]Ph-3,4-dihydrodiol. The (+)-S,S- and (−)-R,R-stereoisomers of B[g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11S,12S-stereoisomer was oxidized at the same rate as the racemate. The 11R,12R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (−)-R,R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11S,12S-dihydrodiol and the B[g]C-11R,12R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (−)-R,R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g]C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH.