Cloning and Characterization of Four Rabbit Aldo-Keto Reductases Featuring Broad Substrate Specificity for Xenobiotic and Endogenous Carbonyl Compounds: Relationship with Multiple Forms of Drug Ketone Reductases

Abstract

Multiple forms of reductases for several drug ketones were isolated from rabbit liver, but their interrelationship and physiologic roles remain unknown. We isolated cDNAs for four aldo-keto reductases (AKR1C30, AKR1C31, AKR1C32, and AKR1C33), which share high amino acid sequence identity with the partial sequences of two rabbit naloxone reductases. The four recombinant enzymes reduced a variety of carbonyl compounds, including endogenous a-dicarbonyls (e.g., isatin and diacetyl), aldehydes (e.g., farnesal and 4-oxo-2-nonenal), and ketosteroids. They differed in specificity for drug ketones and ketosteroids. Although daunorubicin and befunolol were common substrates of all of the enzymes, AKR enzymes specifically reduced naloxone (AKR1C30, AKR1C32, and AKR1C33), metyrapone (AKR1C32 and AKR1C33), loxoprofen (AKR1C31 and AKR1C32), ketotifen (AKR1C30), and naltrexone and fenofibric acid (AKR1C33). AKR1C30 reduced only 17-keto-5b-androstanes, whereas the other enzymes were active toward 3-, 17-, and 20-ketosteroids, and AKR1C33 further reduced 3-keto groups of bile acids and 7a-hydroxy-5b-cholestanes. In addition, AKR1C30, AKR1C31, AKR1C32, and AKR1C33 were selectively inhibited by carbenoxolone, baccharin, phenolphthalein, and zearalenone, respectively. The mRNAs for the four enzymes were ubiquitously expressed in male rabbit tissues, in which highly expressed tissues were the brain, heart, liver, kidney, intestine, colon, and testis (for AKR1C30 and AKR1C31); brain, heart, liver, kidney, testis, lung, and adrenal gland (for AKR1C32); and liver and intestine (for AKR1C33). Thus, the four enzymes correspond to the multiple drug ketone reductases, and may function in the metabolisms of steroids, isatin and reactive carbonyl compounds, and bile acid synthesis.

Probing AKR1C30 and AKR1C31 with Site-Directed Mutagenesis: Identifying the Roles of Residues 54 and 56 in the Binding of Substrates and Inhibitors

“…14) Compared to WT (i.e., AKR1C30), its A54L mutation decreased the sensitivity to valproic acid and phenobarbital, but had no effects on the inhibitory potency of glycyrrhetic acid and carbenoxolone ( Table 2). The results suggest that the residue difference at position 54 between AKR1C30 and AKR1C31 is a major structural factor for the difference in their sensitivity to valproic acid and phenobarbital.…”

“…Site-Directed Mutagenesis Mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, U.S.A.) and the pCold I expression plasmid harboring the cDNA for AKR1C30 14) as the template according to the manufacturer's protocol. The sequences of the forward primers were 5′-GAT GCT GCT TAT CTG TAC CGA AAT GAA-3′ for A54L mutation and 5′-GCT TAT GCG TAC CAA AAT GAA GAG GAA-3′ for R56Q mutation.…”

Identification of a determinant for strict NADP(H)-specificity and high sensitivity to mixed-type steroid inhibitor of rabbit aldo–keto reductase 1C33 by site-directed mutagenesis

“…The reductase and dehydrogenase activities of the enzymes were assayed at 25°C by measuring the rate of change in NAD(P)H absorbance at 340 nm, except that an absorbance at 366 nm was monitored in the assay with high concentrations of NAD(P)H [24]. In the assay of low dehydrogenase activity, the rate of formation in NAD(P)H fluorescence (at 455 nm with an excitation wavelength of 340 nm) was monitored.…”

“…Mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and templates (the pGEX-2T and pCold I expression plasmids harboring the cDNAs for AKR1C33 and AKR1C32, respectively [24]) according to the protocol described by the manufacturer. The mutations were carried out using each set of forward and reverse mutagenic oligonucleotides (Supplementary Table S1).…”

“…They are AKR1C5 (17b/20a-HSD) [7,21], AKRs (1C26, 1C27 and 1C28) acting as 3a/17b-HSDs [22], AKR1C29 (3a/3b/17b/20a-HSD) [23], AKR1C30 (17b-HSD), AKR1C31 (3a/17b/20a-HSD), AKR1C32 (3a/20a-HSD) and AKR1C33 (3a/17b/20a-HSD) [24]. Among the isoforms, AKR1C33 shows the broadest substrate specificity for various steroids including bile acids and 3-keto-5b-cholestanes, and is selectively inhibited by hexestrol, zearalenone and isolithocholic acid [24]. These rabbit isoforms are divided into three groups differing in coenzyme specificity.…”