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 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-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-11 S,12 S-dihydrodiol and the B[g]C-11 R,12 R-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.
Shultz
Carol A
CA
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Palackal
Nisha T
NT
Mangal
Dipti
D
Harvey
Ronald G
RG
Blair
Ian A
IA
Penning
Trevor M
TM
D000409:Alanine / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002911:Chrysenes / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D013058:Mass Spectrometry; D008623:Mercaptoethanol / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010616:Phenanthrenes / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002911:Chrysenes; D007202:Indicators and Reagents; D010616:Phenanthrenes; D011994:Recombinant Proteins; C529145:benzo(g)chrysene-11,12-dihydrodiol; D008623:Mercaptoethanol; C026153:benzo(c)phenanthrene 3,4-dihydrodiol; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D000409:Alanine