Enzymatic Properties of a Member (AKR1C19) of the Aldo-Keto Reductase Family

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) Currently there are more than a hundred known members of this superfamily classified into 14 families.2) The largest family, AKR1, is subdivided into five subfamilies: AKR1A) mammalian aldehyde reductases; AKR1B) mammalian aldose reductases; AKR1C) hydroxysteroid dehydrogenases (HSDs) and prostaglandin F synthases; AKR1D) D 4 -3-ketosteroid-5b-reductases; and AKR1E) mouse keto-reductase. The subfamilies are defined by a Ͼ60% identity in amino acid sequence among subfamily members, but the AKR1C subfamily includes some members that have not been studied as to their enzymatic properties or functions. One such member is AKR1C19 that was found by mouse genomic analysis and cDNA isolation from the liver and gastrointestinal tract. 3) Although AKR1C19 is postulated to be a 3(20)a-HSD-like enzyme based on its highest sequence identity (72%) with human 3(20)a-HSD (AKR1C1) in the AKR1C subfamily, 2) a mouse counterpart of AKR1C1 is shown to be an NADP(H)-dependent 3a/3b/20a-HSD (AKR1C18), 4,5) which shares a lower sequence identity (65%) with AKR1C19. To determine the functional relationship of AKR1C19 with AKR1C18, we examined the enzymatic properties of the recombinant AKR1C19. MATERIALS AND METHODSChemicals Prostaglandins were obtained from Cayman Chemicals (Ann Arbor, MI, U.S.A.), steroids were from Sigma Chemicals and Steraloids (Newport, RI, U.S.A.), and resins for column chromatography were from Amersham Biosciences (Piscataway, NJ, U.S.A.). trans-Benzene dihydrodiol was synthesized by the method of Platt and Oesch. 6) a-and b-3-Hydroxyhexobarbitals (3HBs) were gifts from Dr. R. Takenoshita (University of Fukuoka, Japan). All other chemicals were of the highest grade that could be obtained commercially.cDNA Isolation The cDNA for AKR1C19 was amplified by reverse transcription (RT)-PCR from the total RNA of an 8-week-old male ICR mouse liver. The preparation of the total RNA and RT were carried out as described previously. 7) PCR was performed with Pfu DNA polymerase (Stratagene). The sense primer (5Ј-ATGAGTTCCAAACAGCAA) and antisense primer (5Ј-ACTAAAATTCATCAGAAAAG) correspond to positions 1-18 and 954-973, respectively, of the sequence of a transcript of the AKR1C19 gene. The PCR products of 973 base pairs were ligated into pCR T7/CT-TOPO vectors (Invitrogen), and the expression constructs were transfected into Escherichia coli BL21 (DE3) pLysS according to the protocol described by the manufacturer. The inserts of the cloned cDNAs were sequenced using a CEQ2000XL DNA sequencer (Beckman Coulter) to confirm that the deduced amino acid sequences of the cDNAs are identical to that of AKR1C19 reported by Vergnes et al. 3) Expression and Purification of Recombinant Protein The E. coli cells were cultured in a LB medium containing ampicillin (50 mg/ml) at 37°C until the absorban...

Enzymatic Properties of a Member (AKR1C20) of the Aldo-Keto Reductase Family

“…7,14,15) AKR1C20 also differs from the multiple forms of mouse liver 17b-HSD with respect to the substrate specificity for non-steroidal compounds: While AKR1C20 reduces only a-dicarbonyl compounds with aromatic or alicyclic ring(s), mouse liver 17b-HSD accepts both nonsteroidal alcohols and various carbonyl compounds as the substrates. 14,16) The difference in the properties between AKR1C20 and mouse liver 17b-HSD, together with previous characterization of other mouse enzymes that belong to the AKR1C subfamily, [8][9][10][11][12][13] supports the idea that the three multiple forms of mouse liver 17b-HSD are post-translational products from a gene for AKR1C6. 7) With respect to the dual steroid specificity, AKR1C20 can be classified as a 3a(17b)-HSD (EC 1.1.1.239), of which hamster liver 3a(17b)-HSD has been characterized.…”

“…13) Furthermore, AKR1C20 was inactive towards 9a,11b-prostaglandin F 2 , a good substrate of AKR1C3, 21) and 3-hydroxyhexobarbital, a substrate of mouse AKR1C19. 9) In the reverse reaction, AKR1C20 reduced various 3-ketosteroids with low K m values (Table 2), and exhibited low activities towards 50 mM 5a-and 5b-androstan-3a-ol-17-ones (3 and 4 mU/mg, respectively). The steroid specificity is consistent with that in the forward reaction.…”

Profiling of Embryonic Stem Cell Differentiation

“…Akr1c19 was reported to be highly expressed in the liver and gastrointestinal tract [73]. Aebp2 encodes a zinc finger protein that interacts with the mammalian polycomb repression complex 2 (PRC2) [74].…”

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

“…[6][7][8][9][10] AKR1C12 is also different from NAD(H)-preferring mouse aldo-keto reductases (AKR1C13 and AKR1C19), which do not show significant HSD activity. 11,12) On the other hand, the substrate specificity of AKR1C12 for both steroids and non-steroidal alicyclic alcohols are similar to that of AKR1C24, which has recently been identified as a novel type of NAD(H)-preferring 17b-HSD. 24) The pH optima of the NADH-linked S-camphorquinone reductase and NAD 10.5, respectively, which are also the same as those of AKR1C24.…”

“…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) AKR1C13 11) 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.…”