June 2005 Notes Biol. Pharm. Bull. 28(6) 1075—1078 (2005) 1075 Enzymatic Properties of a Member (AKR1C19) of the Aldo-Keto Reductase Family Shuhei ISHIKURA, Kenji HORIE, Masaharu SANAI, Kengo MATSUMOTO, and Akira HARA* Laboratory of Biochemistry, Gifu Pharmaceutical University; Mitahora-higashi, Gifu 502–8585, Japan. Received January 31, 2005; accepted March 1, 2005 A member (AKR1C19) of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, was shown to be highly expressed in the liver and gastrointestinal tract, but its function remains unknown. In this study, the recombinant AKR1C19 was expressed and purified to homogeneity. The enzyme was a 36-kDa monomer, and reduced a-dicarbonyl compounds such as camphorquinone and isatin using both NADH and NADPH as the coenzymes. Although apparent kinetic constants for the two coenzymes were similar, the NADPH-linked activity was potently inhibited by submillimolar concentrations of NAD؉, but the inhibition of the NADH-linked activity was not significant, suggesting that the enzyme exhibits the NADH-linked reductase activity in vivo. AKR1C19 slowly oxidized 3-hydroxyhexobarbital, S-indan-1-ol and cis-benzene dihydrodiol, but was inactive towards steroids, prostaglandins, monosaccharides, and other xenobiotic alcohols. In addition, the enzyme was inhibited only by dicumarol, lithocholic acid and genistein of various compounds tested. Thus, AKR1C19 possesses properties distinct from other members of the AKR superfamily, and may function as a re- ductase for endogenous isatin and xenobiotic a-dicarbonyl compounds in the liver and gastrointestinal tract. Key words aldo-keto reductase superfamily; AKR1C19; dual coenzyme specificity; isatin; 3-hydroxyhexobarbital dehydroge- nase The aldo-keto reductase (AKR) superfamily is a rapidly Dr. R. Takenoshita (University of Fukuoka, Japan). All other growing group of NAD(P)(H)-dependent oxidoreductases chemicals were of the highest grade that could be obtained that metabolize carbohydrates, steroids, prostaglandins, and commercially. other endogenous aldehydes and ketones, as well as xenobi- cDNA Isolation The cDNA for AKR1C19 was ampli- otic compounds.1) Currently there are more than a hundred fied by reverse transcription (RT)-PCR from the total RNA of known members of this superfamily classified into 14 fami- an 8-week-old male ICR mouse liver. The preparation of the lies.2) The largest family, AKR1, is subdivided into five total RNA and RT were carried out as described previously.7) subfamilies: AKR1A) mammalian aldehyde reductases; PCR was performed with Pfu DNA polymerase (Stratagene). AKR1B) mammalian aldose reductases; AKR1C) hydroxy- The sense primer (5Ј-ATGAGTTCCAAACAGCAA) and steroid dehydrogenases (HSDs) and prostaglandin F syn- antisense primer (5Ј-ACTAAAATTCATCAGAAAAG) cor- thases; AKR1D) D 4-3-ketosteroid-5b-reductases; and respond to positions 1—18 and 954—973, respectively, of AKR1E) mouse keto-reductase. The subfamilies are defined the sequence of a transcript of the AKR1C19 gene. The PCR by a Ͼ60% identity in amino acid sequence among subfam- products of 973 base pairs were ligated into pCR T7/CT- ily members, but the AKR1C subfamily includes some mem- TOPO vectors (Invitrogen), and the expression constructs bers that have not been studied as to their enzymatic proper- were transfected into Escherichia coli BL21 (DE3) pLysS ac- ties or functions. One such member is AKR1C19 that was cording to the protocol described by the manufacturer. The found by mouse genomic analysis and cDNA isolation from inserts of the cloned cDNAs were sequenced using a the liver and gastrointestinal tract.3) Although AKR1C19 is CEQ2000XL DNA sequencer (Beckman Coulter) to confirm postulated to be a 3(20)a-HSD-like enzyme based on its that the deduced amino acid sequences of the cDNAs are highest sequence identity (72%) with human 3(20)a-HSD identical to that of AKR1C19 reported by Vergnes et al.3) (AKR1C1) in the AKR1C subfamily,2) a mouse counterpart Expression and Purification of Recombinant Protein of AKR1C1 is shown to be an NADP(H)-dependent The E. coli cells were cultured in a LB medium containing 3a/3b/20a-HSD (AKR1C18),4,5) which shares a lower se- ampicillin (50 mg/ml) at 37 °C until the absorbance at 600 nm quence identity (65%) with AKR1C19. To determine the reached 0.5. Then isopropyl 1-thio-b-D-galactopyranoside functional relationship of AKR1C19 with AKR1C18, we ex- (1 mM) was added, the culture was continued for 24 h at amined the enzymatic properties of the recombinant 20 °C. The cells were collected and the extract was prepared AKR1C19. as described previously.7) The recombinant AKR1C19 was purified at 4 °C by (NH4)2SO4 fractionation and subsequent MATERIALS AND METHODS three column chromatography steps. The enzyme fraction, precipitated between 35 and 75% (NH4)2SO4 saturation, was Chemicals Prostaglandins were obtained from Cayman dialyzed against Buffer A (10 mM Tris–HCl, pH 7.5, 5 mM 2- Chemicals (Ann Arbor, MI, U.S.A.), steroids were from mercaptoethanol, 1 mM EDTA and 20% glycerol), and was Sigma Chemicals and Steraloids (Newport, RI, U.S.A.), and applied to a Sephadex G-100 column (3ϫ70 cm) equilibrated resins for column chromatography were from Amersham with Buffer A. The enzyme fraction was applied to a Q- Biosciences (Piscataway, NJ, U.S.A.). trans-Benzene dihy- Sepharose column (2ϫ20 cm) equilibrated with Buffer A. 6) drodiol was synthesized by the method of Platt and Oesch. This enzyme eluted with a linear gradient of 0—0.1 M NaCl a- and b-3-Hydroxyhexobarbitals (3HBs) were gifts from was dialyzed against Buffer A, and applied to a Red- ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2005 Pharmaceutical Society of Japan 1076 Vol. 28, No. 6 Sepharose column (1.5ϫ5 cm) equilibrated with the same buffer. The column was washed with Buffer A containing 0.1 M NaCl, and the enzyme was eluted with Buffer A con- ϩ taining 2 mM NAD and 0.1 M NaCl. Assay of Enzyme Activity Reductase and dehydroge- nase activities of AKR1C19 were assayed by measuring the rate of change in NAD(P)H absorbance (at 340 nm) and its fluorescence (at 455 nm with an excitation wavelength of 340 nm), respectively. The standard reaction mixture for the reductase activity consisted of 25 mM Tris–HCl buffer, pH 7.4, 0.1 mM NADH, substrate and enzyme, in a total volume of 2.0 ml. To detect low activities in the cell extract and en- zyme preparations during the purification, 0.1 M potassium phosphate buffer, pH 6.0, was employed instead of the Tris– HCl buffer. Isatin (0.1 mM) was used as the substrate, unless otherwise noted. The dehydrogenase activity was determined ϩ in 25 mM Tris–HCl, pH 7.4, containing 1 mM NAD and an Fig. 1. SDS-PAGE of E. coli Cell Extracts and Purified Recombinant appropriate amount of alcohol substrate. One unit (U) of en- AKR1C19 zyme activity was defined as the amount that catalyzes the The gel (12.5%) was stained with 0.2% Coomassie Brilliant Blue. Lanes: a, molecu- lar mass markers (their positions are indicate in kDa); b, the extract (20 mg) of E. coli reduction or formation of 1 mmol NADH per minute at cells transfected with the expression vector alone; c, the extract (20 mg) of E. coli cells transfected with the expression vector harboring AKR1C19 cDNA; and d, the purified 25 °C. The apparent Km and kcat values were determined over a range of five substrate concentrations at a saturating con- recombinant AKR1C19 (2 mg). centration of coenzyme by fitting the initial velocities to the Michaelis–Menten equation. The kinetic constants and IC50 (inhibitor concentrations required for 50% inhibition) values are expressed as the means of two determinations. Protein concentration was determined by the method of Bradford8) using bovine serum albumin as the standard. RESULTS AND DISCUSSION Purification of Expressed AKR1C19 SDS-PAGE analysis of the E. coli cell extracts (Fig. 1) shows that the cells transfected with the expression plasmids harboring the AKR1C19 cDNA overexpresses a 36-kDa protein, which was not present in the control cells transfected with the vec- tor alone. This cell extract exhibited NADH-linked reductase activity towards isatin (0.0032 U/mg). The recombinant AKR1C19 was purified by both detecting the 36-kDa protein Fig. 2. Effect of NADϩ on NADPH-Linked and NADH-Linked S-Cam- on SDS-PAGE and assaying the isatin reductase activity. The phorquinone Reductase Activities of AKR1C19 The activity was assayed with 0.1 mM NADPH (᭹) or 0.1 mM NADH (᭺) as the enzyme was eluted at a low molecular weight of approxi- ϩ mately 35 kDa on the Sephadex G-100 chromatography step, coenzyme, and is expressed as a percentage relative to the activity without NAD . suggesting its monomeric nature. The final preparation with isatin activity of 0.53 U/mg was greater than 99% pure (Fig. NADP(H). The Km and kcat/Km values for NADH determined 1), and the purification yield was 26% (17 mg/l of cells). in the presence of 0.1 mM S-camphorquinone were 6.9 m M Ϫ1 Ϫ1 pH Dependency The NADH-linked isatin reductase ac- and 3.3 min m M , respectively, and the respective values Ϫ1 Ϫ1 tivity of the purified AKR1C19 was increased by decreasing for NADPH were 11 m M and 2.8 min m M . In the reverse ϩ ϭ the pH from 8.0 to 6.0 in 0.1 M phosphate buffers. The activ- reaction with 2 mM a-3HB as the substrate, NAD (Km ϭ Ϫ1 Ϫ1 ity was also influenced by species and concentrations of the 32 m M and kcat/Km 0.61 min m M ) was a better coenzyme ϩ ϭ ϭ Ϫ1 Ϫ1 assay buffer.