Reduction of Secondary 3-Keto Bile Acids by Aldo-Keto Reductase 1C1 and 1C4.

Abstract Secondary ‘keto' bile acids are produced by the gut microbiome and contain one or more ketones on the steroid core. Plasma concentrations of keto BAs are limited by hepatic reductase activity, leading to hydroxylation of keto BAs. Although the aldo-keto reductase 1 family (AKR1) is implicated, it is not known which enzymes provide this function in the liver. We hypothesized that AKR1C1 and AKR1C4 metabolize 3-keto bile acids. Six bile acids with 3-keto groups were tested as potential substrates using purified, recombinant his₆-tagged AKR1C1-4 and kinetic parameters were determined. AKR1C1 and AKR1C4 were found to exhibit isoform-specific substrate specificity, which may be explained in part by the hydroxylation pattern at carbon 12 of the bile acid core. This may suggest distinct biological roles in mediating bile acid homeostasis in humans. Both enzymes produced only α-OH products, as determined by LC-MS. We further hypothesized that fatty acids would impair reductase activity. AKR1C4 was more susceptible to inhibition compared to AKR1C1, but unsaturated fatty acids, such as linoleic acid, were the most potent inhibitors for both. We observed a 2-10-fold difference in the IC₅₀ of fatty acids for AKR1C4 depending on the tested substrate. Further mechanistic and structure-function studies aim to further characterize the substrate-specific kinetic and inhibition patterns observed and to evaluate the translational impact of AKR activity on plasma bile acid concentrations and cellular signaling.