Substrate Specificity of a Mouse Aldo-Keto Reductase (AKR1C12)

Abstract

The aldo-keto reductase (AKR) superfamily is a rapidly growing group of NAD(P)(H)-dependent oxidoreductases that metabolize carbohydrates, steroids, prostaglandins, and other endogenous aldehydes and ketones, as well as xenobiotic compounds.1,2) Members of this superfamily were classified into 14 families, and each family is subdivided into several subfamilies based on their amino acid sequence similarities. Among the subfamilies, AKR1C is the largest one, which includes mammalian hydroxysteroid dehydrogenases (HSDs), prostaglandin F synthases and related proteins. To date, four members of this subfamily were identified in humans: 20a-HSD (AKR1C1), 3a-HSD type 3 (AKR1C2), 17b-HSD type 5 (also called 3a-HSD type 2 and prostaglandin F synthase; AKR1C3) and 3a-HSD type 1 (AKR1C4).3,4) These members exhibit dihydrodiol dehydrogenase and carbonyl reductase activities for dihydrodiols of aromatic hydrocarbons and xenobiotic carbonyl compounds, and their genes share similar structural organization and are clustered on chromosome 10p15. In mice, a cluster of genes for eight members of the AKR1C subfamily was found on mouse chromosome 13. 5) Three of these mouse members are 20a-HSD (AKR1C18), 6) 3a-HSD (AKR1C14) 7) and 17b-HSD type 5 (AKR1C6), 8) two are dual functional 3a(17b)-HSD (AKR1C20) 9) and 3(17)a-HSD (AKR1C21), 10) and the others are reported to be aldo-keto reductases (AKR1C12, 11) AKR1C1311) and AKR1C19 12)). The five mouse HSDs are NADP(H)-dependent, whereas the three mouse aldo-keto reductases show dual coenzyme specificity for NADPH and NADH or preference for NADH. The expression of the eight mouse enzymes in the tissue differs between themselves. 5)AKR1C12 and AKR1C13 are unique in their high expression in epithelial cells of mouse stomach and have been proposed to be involved in detoxification of xenobiotic carbonyl compounds in the stomach.11) Interestingly, AKR1C12 is identical to a protein (AKRa) that is encoded in an interleukin-3-regulated gene in mouse myeloid cells, suggesting that the enzyme also plays a role in the myeloid cell differentiation. 13)However, there has been only one report on the properties of AKR1C12, in which two xenobiotic carbonyl compounds, 9,10-phenanthrenequinone (PQ) and 4-nitrobenzaldehyde, were found to be good substrates. 11) Thus, the endogenous substrates of the enzyme remain unknown, and its ability to oxidize alcohol substrates has not been studied. In this study, we have examined the coenzyme and substrate specificity of recombinant AKR1C12 in both reduction and oxidation directions at physiological pH in order to elucidate the function related to the high expression levels in the stomach and myeloid cell differentiation. MATERIALS AND METHODScDNA Isolation and Enzyme Purification The cDNA (nucleotides 1-977) for AKR1C12 was amplified from a total RNA of an ICR mouse liver by reverse transcription (RT)-PCR. The preparation of the total RNA and RT were preformed as described previously.14) In the PCR, Pfu DNA polymerase was used together with a sense primer (5Ј-AT-GAG...

Hepatic Xenobiotic Metabolizing Enzyme and Transporter Gene Expression through the Life Stages of the Mouse

“…Akr1c12 and Akr1c13 were down-regulated at 24 months. Akr1c12/13 functions as a dehydrogenase for endogenous hydroxysteroids [27].…”

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…The AKR1C9 is a prototypical 3α-HSD studied in great detail before human enzymes were cloned Bennett et al, 1996;Bennett et al, 1997). The catalytic properties of a number of murine and rat AKR1C enzymes have been described recently Sanai et al, 2007;Matsumoto et al, 2006;Ishikura et al, 2004;Endo et al, 2006). Non-uniformity in the AKR presence in different species imposes limitation on the use of knockout technology for studying the physiological function of the AKRs.…”