Reduction of secondary 3-keto bile acids by aldo-keto reductase 1C1 and 1C4

Secondary "keto" bile acids (BAs) 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 (AKR1) family is implicated, it is not known which enzymes provide this function in the liver. We hypothesized that AKR1C1 and AKR1C4 metabolize 3-keto BAs. Six BAs 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 BA core. This may suggest distinct biological roles in mediating BA homeostasis in humans. Both enzymes produced only α-OH products, as determined by liquid chromatography-mass spectrometry. 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- to 10-fold difference in the IC₅₀ of fatty acids for AKR1C4 depending on the tested substrate. Further mechanistic and structure-function studies aim to characterize the substrate-specific kinetic and inhibition patterns observed and to evaluate the translational impact of AKR activity on plasma BA concentrations and cellular signaling. SIGNIFICANCE STATEMENT: Keto bile acids are bioactive secondary metabolites that are reduced upon enterohepatic recycling to the liver. Here, the substrate specificity, kinetics, and inhibition potential of 2 aldo-keto reductase enzymes, AKR1C1 and AKR1C4, were evaluated. This study suggests that AKR1C1 and AKR1C4 exhibit disparate substrate specificity patterns, reductase activity, and susceptibility to inhibition by fatty acids, which may have broad implications in understanding changes in bile acid homeostasis in metabolic diseases.