Multiplicity of Mammalian Reductases for Xenobiotic Carbonyl Compounds

Drug Metab. Pharmacokinet. 21 (1): 1–18 (2006).

Review Multiplicity of Mammalian Reductases for Xenobiotic Carbonyl Compounds Toshiyuki MATSUNAGA, Shinichi SHINTANI and Akira HARA* Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: A variety of carbonyl compounds are present in foods, environmental pollutants, and drugs. These xenobiotic carbonyl compounds are metabolized into the corresponding alcohols by many mammalian NAD(P)H-dependent reductases, which belong to the short-chain dehydrogenase W reductase (SDR) and aldo-keto reductase superfamilies. Recent genomic analysis, cDNA isolation and characterization of the recombinant enzymes suggested that, in humans, the six members of each of the two superfamilies, i.e., total of 12 enzymes, are involved in the reductive metabolism of xenobiotic carbonyl compounds. They comprise three types of carbonyl reductase, dehydrogenase W reductase (SDR family) member 4, 11b-hydroxysteroid dehydrogenase type 1, L-xylulose reductase, two types of a‰atoxin B1 aldehyde reductase, 20a-hydroxysteroid dehydrogenase, and three types of 3a-hydroxysteroid dehydrogenase. Accumulating data on the human enzymes provide new insights into their roles in cellular and molecular reactions including xenobiotic metabolism. On the other hand, mice and rats lack the gene for a protein corresponding to human 3a-hydroxysteroid dehydrogenase type 3, but instead possess additional ˆve or six genes encoding proteins that are structurally related to human hydroxysteroid dehydrogenases. Characterization of the additional enzymes suggested their involvement in species-speciˆc biological events and species diŠerences in the metabolism of xenobiotic carbonyl compounds.

Key words: Carbonyl reduction; short-chain dehydrogenase W reductase superfamily; aldo-keto reductase superfamily; carbonyl reductase; hydroxysteroid dehydrogenase; species diŠerence alcohols by NADPH-dependent reductases with broad substrate speciˆcity. Some NADPH-dependent reductases reduce quinones through two-electron transfer to the corresponding hydroqinones, which is also mediated by NA(D)PH:quinone reductase. This group of reductases with broad substrate speciˆcity for xenobiotic carbonyl compounds was originally called `aldo-keto or `carbonyl reductases'.2) Subsereductases'1) and W quently, according to the accumulated knowledge on the functions and structures of the reductases, most carbonyl-reducing enzymes have been grouped into two distinct protein families, the short-chain dehydrogenase (SDR)3) and aldo-keto reductase (AKR)4) superfamilies. There had been three excellent reviews on the carbonyl-reducing enzymes by 2000.5–7) Over the recent ˆve years, several new enzymes that may reduce xenobiotic carbonyl compounds have been found on genomic analysis, and thus the enzyme names have been changed. For example, well-known carbonyl reductase (CBR), a member of the SDR superfamily, is now named CBR1 according to the Human Gene Nomencla-

Introduction Aldehydes, ketones and quinones are present in a diverse range of natural and synthetic compounds to which living organisms are exposed. In addition, carbonyl compounds are formed through biological transformation of endogenous components and xenobiotics that are ingested. Aldehydes are chemically reactive and interact with the nucleophilic centers of nucleic acids and proteins. a-Dicarbonyl compounds, such as methyl glyoxal and diacetyl, are more reactive. Ketones are less reactive, and many drugs contain keto group(s). Quinones have a toxic eŠect, i.e., quinoneinduced oxidative stress, when they are reduced through single-electron transfer to the corresponding semiquinones. Organisms have evolved several enzyme systems for detoxifying reactive carbonyl compounds. Such well established pathways include the oxidation of aldehydes to the corresponding carboxylic acids by aldehyde dehydrogenases and aldehyde oxidases, and the reduction of aldehydes and ketones into the corresponding

Received; September 30, 2005, Accepted; October 24, 2005 *To whom correspondence should be addressed : Akira HARA, Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu, Gifu 502-8585, Japan. Tel. & Fax. ＋81-58-237-8586, E-mail: hara＠gifu-pu.ac.jp

1

2

Toshiyuki MATSUNAGA et al. Table 1. Enzymes SDR family CBR1

CBR3 11bHSD 1 DHRS4 L-Xylulose reductase

Gene name (location)

Human enzymes involved in the reduction of xenobiotic carbonyl compounds. Endogenous substrates

Subcellular localization

Accession numbera)

Carbonyl reductase, PG 9-ketoreductase

PG, isatin, ketosteroids Unknown

Cytoplasm Cytoplasm

P16152 O75828

Corticosteroid 11b-dehydrogenase isozyme 1

11-Ketoglucocorticoids

Microsomes

P28845

Retinal

Peroxisomes

Q9BTZ2

DXCR (17q25.3)

Peroxisomal short-chain alcohol dehydrogenase, NADPH-dependent retinol reductase (NDRD) dehydrogenase W L-xylulose reductase, diacetyl Dicarbonyl W reductase

Cytoplasm

Q7Z4W1

AKR7A2 (1p35.1-p36.23) AKR7A3 (1p35.1-p36.23)

A‰atoxin B1 aldehyde reductase member 2, AFAR2 A‰atoxin B1 aldehyde reductase member 3, AFAR1

Succinic semialdehyde

Golgi

O43488

Unknown

Cytoplasm

O95154

AKR1C1 (10 q15-q14) AKR1C2 (10 q15-q14)

20a-HSD, 3(20)a-HSD, DD1

3- and 20-ketosteroids

Cytoplasm

Q04828

3a-HSD type 3, DD2, bile acid-binding protein

3-Ketosteroids

Cytoplasm

P52895

CBR1 (21q22.13) CBR3 (21q22.2) HSD11B1 (1q32-q41) Dhrs4 (14q11.2)

Other names

L-Xylulose,

diacetyl

AKR family

AKR7A2 AKR7A3 AKR1C1 AKR1C2

a)

AKR1C3

AKR1C3 (10 q15-q14)

3a-HSD type 2, 17b-HSD type 5, PGF synthase, DDX

3-, 17- and 20-ketosteroids, PGD2

Cytoplasm

P42330

AKR1C4

AKR1C4 (10 q15-q14)

3a-HSD type 1, DD4, chlordecone reductase

3-ketosteroids

Cytoplasm

P17516

The structural and functional information is available under the accession numbers of UniprotKB W Swiss-Prot (http: WW kr.expasy.org W sprot W ).

ture Committee (HGNC), because of the occurrence of four types of CBR. Several mammalian enzymes that were regarded as CBRs in the previous reviews are now classiˆed into CBR1-CBR3 or have been identiˆed as members of the AKR superfamily. The enzymes that belong to the AKR superfamily have also been named according to the nomenclature for this superfamily. In addition, novel physiological roles of several enzymes have been reported. Therefore, we have reviewed the literature on mammalian carbonyl-reducing enzymes using the new nomenclature in order to clarify their relationship with the previously known enzymes. Carbonyl-reducing Enzymes in the SDR Superfamily The SDR superfamily includes about 3,000 primary structures of functionally heterogeneous proteins, and becomes one of the largest protein families to date.3) The sequence identity of the members of this family is low, but the three-dimensional structures of many b folding patterns members exhibit highly similar a W with a central b-sheet, typical of the Rossmann-fold that participates in cofactor binding. The catalytic tetrad of Asn-Ser-Tyr-Lys is conserved in the members of this superfamiy. Among the members, CBRs, 11b-hydroxysteroid dehydrogenase (HSD) type 1, dehydrogenase W reductase (SDR family) member 4 and L-xylulose

reductase exhibit broad substrate speciˆcities for xenobiotic carbonyl compounds (Tables 1 and 2). CBRs are classiˆed into four types, CBR1, CBR2, CBR3 and CBR4 (Table 3). 1. Carbonyl reductases (CBRs, EC 1.1.1.181) CBR1: The cDNA for the human enzyme was ˆrst cloned by Wermuth et al.,8) who also characterized its properties using the enzyme puriˆed from the brain, and proposed its identity with xenobiotic ketone reductase and prostaglandin (PG) 9-ketoreductase.9) The enzyme is ubiquitously distributed in human tissues.10) The gene is mapped to chromosome 21q22.12, very close to the superoxide dismutase 1 locus at position 21q22.11.11) The mRNA expression in MCF-7 cells is induced 3- or 4-fold in 24 hours by 2,(3)-t-butyl-4-hydroxyanisole, b-naphtho‰avone, or Sudan 1.12) Human CBR1 is a 30 kDa-monomer comprising 277 amino acids, and belongs to the SDR family. Recently, Tanaka et al.13) solved the crystal structure of the enzyme-NADP＋inhibitor complex, which has provided important insights as to the substrate binding site and the design of potent inhibitors of the enzyme, although its tertiary structure is highly similar to that of porcine CBR1.14) Human CBR1 catalyzes the NADPH-dependent reduction of various carbonyl compounds, the best substrates being p- and o-quinones derived from polycyclic

3

Mammalian Carbonyl-Reducing Enzymes Table 2. Enzyme CBR1 CBR3 11bHSD 1

Typical xenobiotic substrates of human reductases.

Drugs

Others

Daunorubicin, doxorubicin, haloperidol, bromperidol, metyrapone, loxoprofen, wortmannin, dolasetron Ketoprofen, metyrapone, insecticidal metyrapone analogues, oracin

DHRS4 L-Xylulose reductase AKR7A2 AKR7A3 AKR1C1

AKR1C2 AKR1C3 AKR1C4

Menadione NNK, menadione, aromatic aldehydes and ketones, 7-ketocholesterol Dicarbonyl compounds with aromatic rings, alkyl phenyl ketones, some aromatic aldehydes and ketones Dicarbonyl compounds, some aromatic aldehydes and ketones

Daunorubicin, ethacrynic acid Dolasetron, naloxone, naltrexone, oxycodone, oracin, befunolol, ketotifen, 10-oxonortriptyline, haloperidol, loxoprofen, acetohexamide, daunorubicin, ethacrynic acid Dolasetron, naloxone, naltrexone, oxycodone, oracin, befunolol, ketoprofen, ketotifen, 10-oxonortriptyline, haloperidol, loxoprofen, acetohexamide, daunorubicin Naloxone, naltrexone Dolasetron, naloxone, naltrexone, oxycodone, oracin, ketoprofen, loxoprofen, acetohexamide, ethacrynic acid, metyrapone, chlordecone

Table 3. Human enzyme

Quinones, aromatic aldehydes, aromatic ketones, NNK

A‰atoxin B1 dialdehyde, dicarbonyl compounds, aromatic aldehydes A‰atoxin B1 dialdehyde, 9,10-phenanthrenequinone, 4-nitrobenzaldehyde Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols 9,10-Phenanthrenequinone, 4-nitrobenzaldehyde, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols

Homologs of human enzymes in other mammalian species.

Mouse Gene name (CH)a)

Enzyme name

Rat Accession (SI)b)

Gene name (CH)a)

CBR1

Cbr1 (16)

CBR1

P48758 (86z)

Cbr1 (11)

*

Cbr2 (11)

CBR2 (Mouse lung CBR) CBR3

P08074

Unknown

Q8K354(85z)

CBR4 11bHSD1 DHRS4 (NDRD) L-Xylulose reductase

NP663570 (86z) P50172 (78z) Q99LB2 (81z)

Cbr3predicted (11) Cbr4 (16) Hsd11b1 (13) Dhr4 (15)

Q91X52 (84z)

Dcxr (Unknown)

AKR7A5 (AFAR2)

Q8CG76 (88z)

RGD:620311 (5)

c)

CBR3

Cbr3 (16)

CBR4 11bHSD1 DHRS4

BC009118 (8) Hsd11b1 (1) Dhrs4 (14)

L-Xylulose reductase AKR7A2

Dcxr (11) Akr7a5 (4)

AKR7A3

Not exist

AKR1C1

Akr1c18 (13)

AKR1C3

Akr1c6 (13)

AKR1C4

Akr1c14 (13)

Akr7a3 (5) AKR1C18 (20a-HSD) AKR1C6 (17b-HSD type 5) AKR1C14 (3a-HSD)

Q8K023 (68z)

LOC171516 (17)

P70694 (69z)

LOC364773 (17)

Q91WT7 (67z)

LOC191574 (17)

Enzyme name

Accession (SI)b)

CBR1 (Non-inducible CBR)

P47727 (85z)

CBR3

XP22164 (85z)

CBR4 11bHSD1 DHRS4 (NDRD) L-Xylulose reductase

Q7TS56 (84z) P16232 (76z) Q8VID1 (81z)

AKR7A4 (AFAR2) AKR7A1 (AFAR1) AKR1C8 (20a-HSD) RAKh (17b-HSD type 5) AKR1C9 (3a-HSD)

Q920P0 (84z) Q8CG45 (87z) P38918 (81z) P51652 (71z) NP001014240 (73z) P23457 (69z)

a)

Most rat genes are named tentatively. CH, chromosome. Accession: The number in UniprotKB W Swiss-Prot or amino acid sequence in NCBI database. SI: sequence identity with the relevant human enzyme. c) The CBR2 gene does not exist in the human genome. b)

aromatic hydrocarbons.9,15) In human liver and placenta, CBR1 acts as a major quinone reductase, but the enzymatic reduction of several o-quinones results in redox cycling of the quinones, leading to the generation of semiquinones and the superoxide anion.16) Thus,

CBR1 seems to be involved in both the detoxiˆcation and intoxication of quinones. The endogenous substrates of CBR1 are suggested to be PGs, some 3ketosteroids and isatin, of which isatin is the best one, showing a high a‹nity and turnover number that are

4

Toshiyuki MATSUNAGA et al.

compatible with those of xenobiotic o-quinones.17) In addition, CBR1 catalyzes the reduction of the 4-keto, aldehyde and C＝C of 4-oxonon-2-enal, a product of lipid peroxidation.18) CBR1 homozygous null mice are nonviable,19) suggesting that the enzyme plays a nonredundant role in cell signaling during embryogenesis and development. A study on the biological function of CBR1 in A549 adenocarcinoma cells suggested that the enzyme is involved in serum-free-induced apoptosis in the cells.13) On the other hand, several studies involving tumor tissue specimens have indicated that a decrease in CBR1 expression is correlated with the degree of dediŠerentiation in hepatocellular carcinomas,20) poor survival and lymph node metastasis in epithelial ovarian cancer,21) and tumor progression and angiogenesis in lung cancer.22) Therefore, elucidation of the mechanisms underlying the suggested roles of the enzyme in cell signaling, apoptosis and cancer progression is an important future goal. Human CBR1 reduces several therapeutic agents and toxicologically important compounds (Table 2). The drug substrates include daunorubicin and doxorubicin,6) loxoprofen,23) metyrapone,23) haloperidol,24) bromperidol,25) timiperone,26) and wortmannin,27) which has been proposed to be a potential antineoplastic agent. Since daunorubicinol, the reduced product of antiproliferative daunorubicin, is cardiotoxic, CBR1 is thought to be responsible for the severe cardiotoxicity associated with daunorubicin treatment.6) This is supported by the ˆnding that mice heterozygous for a null allele of CBR1 show reduced sensitivity to anthracycline-induced cardiotoxicity.19) The enzyme also reduces 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), a tobacco-speciˆc potent carcinogen, into (S)-4-methylnitrosamino-1-(3-pyridyl)-1-butanol, which is more tumorigenic. The signiˆcance of the NNK metabolism by CBR1 and other reductases was described in a recent review by Maser and BreyerPfaŠ.28) Other animal enzymes that have similar sequences to human CBR1 are classiˆed as CBR1s. The genes for CBR1s of mouse and rat have been identiˆed through genomic analyses (Table 3). In the mouse genome, an additional gene (accession no.: LOC435489) encoding a protein exhibiting 87z sequence identity with mouse CBR1 is predicted. Similarly, two cDNAs for gonadotropin-inducible and non-inducible CBR1 isoforms (86z sequence identity) have been isolated from rat ovary.29) The inducible rat CBR is encoded by the genomic CBR1 gene, and identical to a testicular CBR that was previously characterized.30) The noninducible isoform might correspond to another form of the two rat testicular CBRs,31) although the gene for this isoform was not found on the present genomic sequencing. Two cDNAs for such CBR1 isoforms have also

been isolated from rabbit liver32) and Chinese hamster ovary cells.33) Thus, these animal species probably have two genes for CBR1 isoforms, in contrast to the existence of only one gene for human CBR1. The substrate speciˆcities of the CBR1s of these animals and pig34) are essentially identical to that of the human enzyme, except that pig CBR1 exhibits 20b-HSD activity. Mouse and Chinese hamster CBR1s, similar to the human enzyme, are distributed in many tissues,33,35) whereas the rat enzyme is speciˆcally expressed in reproductive tissues and the adrenal gland.31) CBR2: This enzyme is not present in human tissues, as its gene has not been found in the human genome. However, CBR2 is highly expressed in the mitochondria of epithelial cells of mouse, guinea-pig and pig lungs.36–38) In addition, mitochondrial CBR2, which was previously known as a sperm protein, P26h, is present in testis and epididymis of hamster.39) As a gene for CBR2 is not found in the rat genome (Table 3), CBR2 protein has not been detected in rat tissues. CBR2s are homotetramers composed of 26 kDa-subunits, and exhibit low sequence identify with monomeric CBR1. The enzymes that reduce various aliphatic, alicyclic and aromatic carbonyl compounds with lower Km values are activated by fatty acids and inhibited by pyrazole, a known inhibitor of alcohol dehydrogenase. The diŠerences between hamster CBR2 and the lung enzymes of other species are in the cofactor speciˆcity and the reversibility of the reaction. The hamster enzyme shows a cofactor preference for NAD(H) and e‹ciently catalyzes the oxidation of 5a-dihydrosterosterone, whereas the lung enzymes utilize NADP(H) as a preferred cofactor and do not exhibit signiˆcant dehydrogenase activity. The tissue distribution and catalytic properties suggest that lung CBR2s function in the detoxication of carbonyl compounds derived through lipid peroxidation and xenobiotic carbonyl compounds ingested from the airways and blood.36–38) The hamster CBR2 is thought to act as a 3a-HSD to control the intracellular concentration of a potent androgen, 5a-dihydrosterosterone, during spermatogenesis.39) A crystallographic study of mouse CBR2 has provided insights into its unique tetrameric structure and cofactor binding mode.40) The key residues in the cofactor speciˆcity were also identiˆed by site-directed mutagenesis studies of mouse and hamster CBR2s.39,41) CBR3: The gene for this enzyme was ˆrst identiˆed 62 kilobases apart from the CBR1 gene on chromosome 21.42) The encoded protein is composed of 277 amino acids, and its sequence identity with human CBR1 is 71z and that with animal CBR2s is less than 23z. Recently, a natural allelic variant of CBR3 (V244M) was found, and the kinetic constants for NADPH and menadione of the recombinant CBR3 and V244M variant were determined.43) The mRNA for CBR3 is

Mammalian Carbonyl-Reducing Enzymes

suggested to be expressed in various tissues,33) although the expression level is much lower than that of CBR1 (personal communication from Dr. T. Terada). cDNA microarray analysis in patients with keloid showed that the CBR3 gene, together with eight other genes, in keloid tissue is consistently upregulated.44) The CBR3 gene has been identiˆed or predicted in the mouse and rat genomes (Table 3). In Chinese hamster, CBR3 (86z sequence identity with human CBR3) exhibits high activity toward daunorubicin and isatin compared to CBR1, but conversely lacks oxidoreductase activity toward PGs, which are substrates for CBR1.33) The mRNA for CBR3 is detected in the order of kidneyÀbrainÀliver in Chinese hamster, which diŠers from the expression patterns of the mRNAs for CBR1 and its isoform. Thus, reports on CBR3 have been few, and the concentrations of CBR3 proteins in human and animal tissues are unknown. Further studies on the enzymology and gene regulation of CBR3 are necessary to elucidate its roles in the metabolism of endogenous and exogenous compounds, and in the pathogeneses of diseases. CBR4: The gene for this enzyme was predicted on recent genomic analyses, and is located on human chromosome 4 (4q32.3). The encoded protein (accession no. Q8N4T8) is composed of 237 amino acids, and exhibits low sequence identity (º25z) with other types of CBR. The CBR4 gene is present in laboratory animals (Table 3) and dog (accession no.: XP534547), suggesting a role in the metabolism of endogenous compounds for CBR4. Although the enzyme is named carbonyl or carbonic reductase 4, its enzymatic properties and tissue distribution remain unknown. 2. 11b-HSD type 1 (HSD11B1, EC 1.1.1.146) 11b-HSD is a microsomal protein that catalyzes the interconversion of active cortisol into inactive cortisone. Two isoforms, NADP(H)-dependent HSD11B1 and NAD(H)-dependent 11b-HSD type 2 (HSD11B2), in mammalian tissues have been cloned and characterized. HSD11B1 is expressed in a wide range of tissues, acts predominantly as a reductase in intact cells and tissues by regenerating active cortisol from cortisone, and regulates glucocorticoid access to the glucocorticoid receptor. HSD11B2 is mainly expressed in mineralocorticoid target tissues such as kidney and colon, acts only as a dehydrogenase by producing inactive cortisone, and protects the mineralocorticoid receptor from high levels of receptor-active cortisol. The roles and enzymology of the two isoforms were described in recent reviews.45,46) Increases in the activity and expression of HSD11B1 have been implicated in the pathogeneses of many common conditions including obesity, insulin resistance, the metabolic syndrome, the polycystic ovarian syndrome, osteoporosis and glaucoma.47) Therefore, selective HSD11B1 inhibition has been

5

proposed as a novel therapeutic strategy for many of these conditions. Mammalian HSD11B1s exhibit high sequence identity, and the crystal structures of guineapig and mouse HSD11B1s were recently solved.48,49) Selective and potent HSD11B1 inhibitors will be developed in the near future through structure-based drug design using the crystal structures. HSD11B1 also plays a role in the metabolism of non-glucocorticoid carbonyl compounds, i.e., oracin, ketoprofen, metyrapone, insecticidal metyrapone analogues, NNK, menadione, and some aromatic aldehydes and ketones.50–52) On the reduction of NNK, the human enzyme forms both the (S)- and (R)-enantiomers of 4-methylnitrosamino-1-(3-pyridyl)-1-butanol. This enantioselectivity is in contrast to those of human CBR1 and other AKR1C isoforms, which form the (S)-enantiomer on the reduction.28) Recently, HSD11B1 was reported to e‹ciently catalyze the reduction of 7ketocholesterol, the major dietary oxysterol, into 7hydroxycholesterol, species-speciˆc diŠerences in the stereospeciˆc reduction being observed between the rat, human, and hamster enzymes.53) The human and rat enzymes reduce 7-ketoxycholesterol into 7b-hydroxycholesterol, whereas the hamster enzyme catalyzes the interconversion of 7-ketoxycholesterol into both 7aand 7b-hydroxycholesterol. Although mammalian HSD11B1s show similar substrate speciˆcities for xenobiotic compounds, the stereoselective reduction or the may diŠer depending with the species and W reduced compound. reductase (SDR family) member 3. Dehydrogenase W 4 (DHRS4, EC 1.1.1.-) The cDNA for human DHRS4 was ˆrst cloned as a peroxisomal 2,4-dienoyl-CoA reductase-related protein.54) DHRS4 belongs to the SDR family, and has a C-terminal peroxisomal targeting signal 1 (Ser-ArgLeu). It has also been called SDR-SRL, peroxisomal short-chain dehydrogenase and NADPH-dependent reductase (NDRD), because the retinol dehydrogenase W rabbit and pig homologues of human DHRS4 exhibit NADPH-linked retinal reductase activity.55) In addition, two alternative splicing forms of human DHRS4, SDRSRL1 (lacking residues 118–204) and SDR-SRL-2 (lacking 85–204), have been isolated.54) DHRS4, SDRSRL1 and SDR-SRL-2 are now designated as DHRS4 isoforms 1, 2 and 3, respectively. Since there has been no literature on the properties of human DHRS4, we characterized the recombinant DHRS4 isoform 1. It is an NADPH-dependent tetrameric reductase for xenobiotic carbonyl compounds, but diŠers from the enzymes of other species (described below) in its low activity toward retinoids and instability at low temperature. The human enzyme reduces all-trans-retinal with a Km (0.8 units W mg W mM), and does not exhibit low Vmax W signiˆcant 9-cis-retinol reductase or all-trans-retinol

6

Toshiyuki MATSUNAGA et al.

dehydrogenase activity. The enzyme is gradually inactivated at 49 C, in contrast to the stable enzymes of other species. The cold inactivation might have prevented detection of the enzyme activity in human tissues in previous studies on CBRs. The mRNA for DHRS4 isoform 1 is ubiquitously expressed in human tissues, in which expression of the mRNA for the isoform 2 is low. No enzymatic activity is observed for recombinant isoform 2. Thus, the physiological role of human DHRS4 is not clear, but the enzyme reduces alkyl phenyl ketones and might be involved in the metabolism of the butyrophenone type of drugs. The cDNAs for other mammalian DHRS4s have been cloned and their genes are shown in Table 3. The rabbit and pig DHRS4s are peroxisomal homotetramers, being composed of 27-kDa subunits.55) The enzymes reduce alkyl phenyl ketones, a-dicarbonyl compounds, retinals and some aromatic aldehydes using NADPH as a cofactor, and are identical to a tetrameric CBR that was previously puriˆed from rabbit heart.56) Of these xenobiotic substrates, 9,10-phenanthrenequinone is e‹ciently reduced by the enzymes, and the reduction results in the formation of reactive oxygen species (ROS).57) The enzymes show high catalytic e‹ciency for all-trans-retinal and 9-cis-retinal (the Vmax W Km values of the pig enzyme are 618 and 30 units W mg W mM, respectively, at 259C and pH 7.4), and oxidize all-trans-retinol with a low Vmax W Km value of 1.5 units W mg W mM.55) Mouse DHRS4 also exhibits high reactivity towards retinoids, and its content in the liver increases with cloˆbrate feeding.58) In the three animals, mRNA for DHRS4 is expressed in many tissues, of which liver and kidney exhibit the highest expression. We have cloned a cDNA for DHRS4 from dog liver, and characterization of the recombinant enzyme revealed that it is identical to a high-molecular weight CBR that is highly expressed in the liver.59) 4. L-Xylulose reductase (EC 1.1.1.10) L-Xylulose reductase catalyzes the NADPHdependent reduction of L-xylulose to xylitol in the uronate cycle of glucose metabolism. The human, mouse, rat, guinea pig and hamster enzymes have been cloned and shown to be identical with diacetyl reductase (EC 1.1.1.5) that reduces various a-dicarbonyl compounds including o-quinones, and aromatic aldehydes and ketones.60,61) Because of its broad substrate speciˆcity, L-xylulose reductase is thought to be involved in the metabolism of xenobiotic carbonyl compounds, although drugs containing carbonyl groups have not been examined as substrates. The enzyme is a homotetramer, and exhibits high sequence identity of 65z with mouse CBR2. The crystal structure of human 62) L-xylulose reductase is similar to that of mouse CBR2, but there is a signiˆcant diŠerence in their substrate binding clefts. L-Xylulose reductase also diŠers from

CBR2 in inhibitor sensitivity and tissue distribution. reductase is inhibited by n-butyric acid, but not by pyrazole. The enzyme is distributed in many tissues, of which the liver and kidney show high expression of the enzyme. Furthermore, the L-xylulose reductases of mouse, rat, guinea pig and hamster are rapidly dissociated into their inactive dimeric forms at low temperature.61,63) L-Xylulose

Carbonyl-reducing Enzymes in the AKR Superfamily The 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.4) Currently there are more than a hundred known members of this superfamily that is classiˆed into 14 families. The nomenclature system is similar to that for the cytochrome P450 superfamily, but, unlike that system, it involves amino acid sequence comparisons. Within a given family, subfamilies are deˆned according to À60z identity in amino acid sequence among subfamily members. The largest family, AKR1, is subdivided into ˆve subfamilies: AKR1A, mammalian aldehyde reductases; AKR1B, mammalian aldose reductases; AKR1C, HSDs and PGF synthases; AKR1D, D4-3-ketosteroid-5b-reductases; and AKR1E, mouse keto-reductase. More information on this family is available on the AKR superfamily homepage akr). Among the enzymes in this (www.med.upenn.edu W superfamily, aldose reductases, aldehyde reductases, HSDs, PGF synthases and a‰atoxin B1 reductases (AFARs, belonging to the AKR7A subfamily) exhibit broad substrate speciˆcities for xenobiotic carbonyl compounds. In this section, we describe recent studies on the characteristics of HSDs, PGF synthases and AFARs, focusing mainly on their roles in xenobiotic metabolism. Aldehyde and aldose reductases are not included in this section, because they are not involved in the reduction of drug ketones, except for daunorubicin and acetohexamide.23) A recent review described the properties and functions of aldose reductase and closely related aldo-keto reductases,64) of which AKR1B10, i.e., human aldose reductase-like protein, has been recognized as a new diagnostic marker of hepatocellular carcinomas65) and smokers' non-small cell lung carcinomas.66) Since the mouse and rat genomes contain many genes for the enzymes belonging to the AKR1C subfamily compared with the human genome, we divide the enzymes into human and rodent ones in order to avoid more confusion. 1. Enzymes in the AKR7A subfamily AFAR catalyzes the NADPH-dependent reduction of a‰atoxin B1 dialdehyde, a toxic metabolite produced from a‰atoxin B1 by CYP3A, into unreactive monoand di-alcohol derivatives.67) The human, mouse and rat

Mammalian Carbonyl-Reducing Enzymes

enzymes have been cloned and characterized, and their genes are shown in Tables 1 and 3. The enzymes are dimers, and show similar broad substrate speciˆcities for various aromatic aldehydes and dicarbonyl compounds. Of these substrates, 2-carboxybenzaldehyde is useful as a diagnostic model substrate for AFAR, because it is an inactive or poor substrate for other reductases in the SDR and AKR families.68) In human and rat tissues, AFAR is present in two isoforms, which have been classiˆed into two classes, AFAR1 and AFAR2, with respect to sequence similarity, a‹nity for succinic semialdehyde, intracellular localization and inducibility by xenobiotics. AFAR1: This class of AFAR includes human AKR7A369) and rat AKR7A1,70) the subunits of which are composed of 327 amino acids and exhibit 81z sequence identity. The rat enzyme reduces various aldehydes, but its Km value for succinic semialdehyde, an endogenous substrate, is high.71) The enzyme is expressed in liver and several extrahepatic tissues, and is induced by phenolic antioxidants, ethoxyquin, coumarin, and dietary indoles and isothiocyanates.70,72,73) Modulation of the level of AKR7A1 is also suggested to correlate with the carcinogenesis caused by a‰atoxin B1.70,72) In rat liver, AKR7A1 exists as both a homodimer and a heterodimer with a subunit of AKR7A4 belonging to another class of AFAR. The crystal structure of AKR7A1 revealed the details of the dimeric structure and substrate recognition.74,75) Little is known about the catalytic activity and inducibility of human AKR7A3, which is reported to reduce some substrates (Table 2).69) AFAR2: This class includes human AKR7A2,76) mouse AKR7A5,77) and rat AKR7A4,78) the subunits of which are composed of 359–367 amino acids and exhibit high sequence identity (À87z) with each other. The enzymes are constitutively expressed in many tissues, in which they are thought to be associated with the Golgi apparatus.79) Their substrate speciˆcities are similar to that of AFAR1, but AFAR2 shows a low Km value for succinic semialdehyde.68,76–79) Human AKR7A2 is indeed identical to succinic semialdehyde reductase,80) indicating its physiological role in g-hydroxybutyate synthesis. Human AKR7A2 reduces various xenobiotic aromatic aldehydes and a-dicarbonyl compounds, but is essentially inactive towards aliphatic and aromatic ketones.68,76) Although the human enzyme reduces daunorubicin and ethacrynic acid at low rates,68) other drugs have not been examined as substrates. 2. Human enzymes in the AKR1C subfamily Four human enzymes, AKR1C1-AKR1C4, have been cloned and characterized. The genes exist in a cluster on chromosome 10 (Table 1). Although two genes, AKR1CL1 and AKR1CL2, for AKR1C-like proteins have been found near the cluster, their gene functions are unknown: The AKR1CL1 gene codes for a

7

hypothetical polypeptide of 129-amino acids (accession no.: Q6ZN81), and the AKR1CL2 gene encodes a monomeric protein of 320-amino acids (accession no.: Q9BU71) that exhibits low reductase activity only toward 9,10-phenanthrenequinone.81) AKR1C1AKR1C4 are NADP(H)-dependent monomeric dehydrogenases composed of 323 amino acids and exhibit high sequence identity (À83z), but their biochemical properties are diŠerent. The enzymes are 3a-, 17b- and W or 20a-HSDs, and play roles in the pre-receptor regulation of steroid receptors, nuclear orphan receptors and W or membrane-bound ligand-gated ion channels. The enzymes are suggested to be targets for the treatment of prostate cancer, breast cancer, endometriosis and endometrial cancer.82) The other characteristic of the enzymes diŠering from those of the members of the AKR superfamily is that they show high dihydrodiol dehydrogenase activity (DD, EC 1.3.1.20), which oxidizes the trans-dihydrodiols of aromatic hydrocarbons into the corresponding catechols. Therefore, AKR1C1, AKR1C2, AKR1C3 and AKR1C4 are called DD1, DD2, DDX and DD4, respectively. DD was ˆrst shown to be a detoxiˆcation enzyme in the metabolism of carcinogenic polycyclic aromatic hydrocarbons, but has been regarded as a toxication enzyme, because oxidation by the enzyme yields reactive and redox-active o-quinones and ROS.83) In addition, AKR1C1-AKR1C4 metabolize PGs and a number of xenobiotic compounds. In this section, we summarize the main characteristics of the individual enzymes and describe their roles in xenobiotic carbonyl metabolism. AKR1C1: This enzyme is known as 20a-HSD (EC 1.1.1.149), and is distributed in many tissues.84–86) AKR1C1 exhibits additional 3a- and 3b-HSD activities depending on the steroid structure: It reduces 3-keto-5bdihydrosteroids into the 3a-hydroxy derivatives, whereas 3b-hydroxy products are formed on the reduction of 3-keto-5a-dihydrosteroids.87,88) The 5aandrostane-3b,17b-diol and 5a,3b-tetrahydroxypregnanes produced are ligands of estrogen receptor b89) and antagonists for the g-aminobutyric acid type A (GABAA) receptor,90,91) respectively. Thus, the enzyme plays important roles in the steroid metabolism through the formation of the above steroids as well as inactivation of progesterone, neuroactive 5a W b-pregnan-3a-ol20-ones (positive modulators of the GABAA receptor), and 5a-dihydrotestosterone.82) AKR1C1 diŠers from AKR1C2 (3a-HSD type 3) by only seven amino acids, of which the residue at position 54 (Leu in AKR1C1 and Val in AKR1C2) has been demonstrated to be a determinant of the steroid speciˆcity of the two enzymes in site-directed mutagenesis and crystallographic studies.88,92,93) AKR1C1 reduces a variety of non-steroid carbonyl compounds including PGs and NNK. The enzyme

8

Toshiyuki MATSUNAGA et al. Table 4. Substrate NNK Metyrapone R-Ketoifen S-Ketotifen Dolasetron Naloxone Naltrexone Oxycodone

Comparison of kinetic constants for xenobiotic substrates among human enzymes.

CBR1

HSD11B1

Km

Vmax W Km

Km

7 0.9

400 534

12

NS NS NS NS NS NS

AKR1C1

Vmax W Km

Km

Vmax W Km

20

0.2

90 NS 500 1840 3280 41 59 17

ND NT NT NT NT NT NT

0.01 0.05 0.06 0.4 0.4 1.8

AKR1C2 Km

0.3 NS 0.008 0.004 0.03 0.02 0.1 NS

AKR1C4

Vmax W Km

57 7250 25000 900 570 210

Km

8 19

0.2 0.006 0.01 0.4

Vmax W Km

10 35 NS NS 2570 12400 6440 79

Km＝mM and Vmax W Km＝units W mg W M (1 unit＝nmol of reduced product or NADPH W min). ND: the activity was detected, but the kinetic constants have not been determined. NS: no signiˆcant activity was detected. NT: not tested as a substrate. The values for naloxone, naltlexone and oxycodone were determined at pH 7.4 and 379C.

reduces a variety of xenobiotic carbonyl compounds, but diŠers from the AKR7A enzymes in its ability to reduce aromatic ketones.68) It exhibits low PGF synthase activity.85,94) On the reduction of NNK, the enzyme shows the lowest Km value of all known NNK reductases (Table 4).28) Drug substrates, which have been identiˆed using the puriˆed hepatic and recombinant enzymes,23,24,95,96) are listed in Table 2. Although these drugs are not speciˆc substrates for AKR1C1, catalytic e‹ciency comparison indicated that dolasetron, an antiemeric 5-HT3 receptor antagonist, is most e‹ciently reduced by the enzyme.95) The Z- and E-10ketonortriptylines are also good substrates for this enzyme.24) AKR1C1 is selectively and potently inhibited by benzbromarone,97) suggesting a possible drug-drug interaction between this drug and the above drug substrates of the enzyme. AKR1C1 is induced by ethacrynic acid, polycyclic aromatic hydrocarbons, other polycyclic aromatic compounds, electrophilic Michael acceptors, phenolic antioxidants, ROS, 4-hydroxynonenal, and dietary indoles and isothiocyanates.73,98–100) The induction by the polycyclic aromatic hydrocarbons supports the role of the enzyme in the activation of polycyclic aromatic hydrocarbons,83) whereas the enzyme provides an inducible cytosolic barrier to 4-hydoxynonenal in oxidative stress by metabolizing the toxic aldehydes.100) The inducers include dietary indoles and isothiocyanates, which may in‰uence the metabolism of steroids and drugs catalyzed by AKR1C1. AKR1C2: This enzyme is the type 3 isozyme of human 3a-HSD (EC 1.1.1.213), and its mRNA is detected in many tissues.84–86) Despite its structural similarity with AKR1C1, this enzyme mainly exhibits 3a-HSD activity. The enzyme is believed to play important roles in the inactivation of 5a-dihydrotestosteb-pregnan-3arone and the synthesis of neuroactive 5a W ol-20-ones from their precursors.82) In addition to the

roles of AKR1C2 in the development and progression of prostate cancer,82) recent literature suggested possible involvement of the enzyme in the pathogenesis of glaucoma and obesity in women. Signiˆcantly high expression and activity of AKR1C2 are observed in human glaucomatous optic nerve head astrocytes,101) and the expression and activities of AKR1C2 and AKR1C1 in omental adipose tissue are positive correlates with adiposity in women.102) The substrate speciˆcity of AKR1C2 for non-steroid carbonyl compounds85,103) is similar to that of AKR1C1 (Table 2), but AKR1C2 is inactive towards ethacrynic acid and metyrapone.23) The substrate speciˆcity for drugs also overlaps those of the other enzymes in the SDR and AKR superfamilies (Table 4), and AKR1C2 may be the major enzyme in the reductive metabolism of ketotifen because of its extremely high reactivity towards both the R- and S-forms of the drug.24) There has been no report on the induction of AKR1C2 by xenobiotics. The enzyme is highly inhibited by bile acids, and is identical to a human bile acid-binding protein.103,104) AKR1C3: This enzyme exhibits a broad tissue distribution.84–86) Although its tissue expression can be detected by means of immunochemical methods105) and ampliˆcation of the cDNA, AKR1C3 has not been puriˆed from human tissues, in contrast to the puriˆcation of other AKR1C enzymes.23,24,28,103,106) The inability to purify the enzyme from the tissues may result from the high liability of the enzyme in tissue homogenates and cell lysates.107) AKR1C3 was ˆrst named 3a-HSD type 2,108) but exhibits high 17b-HSD activity toward 4-androstene-3,17-dione, so it is also classiˆed as 17b-HSD type 5 according to the nomenclature for 17bHSD isozymes (EC 1.1.1.62).109) In addition, AKR1C3 is identical to PGF synthase (EC 1.1.1.188) that catalyzes both the formation of PGF2a from PGH2, and the interconversion between PGD2 and 9a,11b-PGF2.110,111)

Mammalian Carbonyl-Reducing Enzymes

Thus, the enzyme is involved in the metabolism of androgens and PGs, and is suggested to be involved in the development of prostate cancer by forming testosterone and the diŠerentiation of leukemia cells caused by metabolizing PGD2.82,112) AKR1C13 is signiˆcantly up-regulated during the diŠerentiation of HL-60 cells by all-trans-retinoic acid and vitamin D3,113) and its mRNA expression in human ˆbroblasts is also induced by thyroid hormone.114) Recently, three crystal structures of the enzyme were determined to elucidate the mechanism underlying the multiple speciˆcity and to develop speciˆc inhibitors.115–117) Such several inhibitors selective for AKR1C1, AKR1C2 and AKR1C3 have been reported.118) Only 9,10-phenanthrenequinone and 4-nitrobenzaldehyde have been reported to be xenobiotic substrates of AKR1C3.85,111) We tested several drugs as substrates, and found that naloxone and naltrexone are reduced by the enzyme (unpublished results). The Km and Vmax W Km values for naloxone are 1.3 mM and 2.6 units W mg W M, respectively, the respective values for naltrexone being mg W M at 379 C and pH 7.4. 0.6 mM and 54 units W Comparison of these values with those for other enzymes (Table 4) suggests that AKR1C3 is not a major enzyme in drug metabolism. AKR1C4: This enzyme is 3a-HSD type 1, and exhibits higher a‹nity and velocities for most 3-keto and 3a-hydroxysteroids than the other types of the enzyme do. It also exhibits low 20a-HSD and 17b-HSD activities,86) and is inhibited by phenolphthalein and steroidal anti-in‰ammatory agents.97) Crystallization of AKR1C4 has not been achieved. This enzyme is expressed speciˆcally in the liver, and is involved in the catabolism of circulating steroid hormones and the metabolism of bile acids.82,119) The liver-speciˆc expression of AKR1C4 mRNA is regulated by transcripg, tion factors, hepatocyte nuclear factor (HNF)-4 a W HNF-1a and variant HNF-1.120,121) As AKR1C4 was ˆrst cloned as a cDNA for chlordecone (an insecticide) reductase,122) it reduces various xenobiotic carbonyl compounds and drugs (Table 2). AKR1C4 e‹ciently reduces naloxone, naltrexone and oxycodone, although it is almost inactive towards befunolol, daunorubicin, haroperidol and ketotifen (Table 4). The enzyme probably plays an important role in the reduction of these three drugs and chlordecone. On the other hand, several drugs such as sulfobromophthalein,123) cloˆbric acid derivatives124) and anti-in‰ammatory 2-arylpropionic acids125) enhance the enzyme activity of AKR1C4. While there has been no report on the induction of AKR1C4 by xenobiotics so far, an interindividual diŠerence in hepatic chlordecone reductase activity has been reported.126) A large interindividual diŠerence has also been noted in the hepatic expression of AKR1C4 mRNA, which is related to the

9

diŠerences in the amounts of HNF-1a, HNF-4a and HNF-4g.127) In addition, a variant of AKR1C4 (S145C W L311V) that decreases the enzyme activity has been identiˆed.128) Such studies on interindividual diŠerence are needed for the other AKR1C enzymes, because they are involved not only in the metabolism of other drugs, but also in several diseases. 3. Rodent enzymes in the AKR1C subfamily NADP(H)-dependent 20a-, 17b- and 3a-HSDs, which correspond to human AKR1C1, AKR1C3 and AKR1C4, respectively, have been cloned and characterized from mouse and rat tissues (Table 3). The rodent enzymes exhibit the following diŠerences in substrate speciˆcity and tissue distribution from the relevant human enzymes, although data on rat aldo-keto reductase h (RAKh) have not been published. (1) Mouse AKR1C1888,129) and rat AKR1C8130) are 20a-HSDs, exhibiting additional 3a W 3b-HSD activity that is observed with human AKR1C1. In contrast to the high DD activity of human AKR1C1, the rodent enzymes show low DD activity, and signiˆcantly high reductase activity toward nonsteroidal substrates such as a-dicarbonyl compounds, aromatic aldehydes and ketones.88) In addition, the tissue distributions of the rodent enzymes diŠer from that of the ubiquitous human enzyme. Mouse AKR1C18 is highly expressed in the ovary and kidney, and at lower levels in many other tissues.88,131) AKR1C8 is ovary-speciˆc in female rats130) and its expression in male rat tissues is quite low. (2) Mouse AKR1C6132) and rat RAKh are 17b-HSDs that are associated with low 20a-HSD activity, and high DD and reductase activities toward a variety of nonsteroidal carbonyl compounds. Thus, they appear to be type 5 isozymes of 17b-HSD that correspond to human AKR1C3. However, the rodent enzymes do not exhibit 3a-HSD and PGF synthase activities, which are high for AKR1C3. AKR1C6 is a liver-speciˆc enzyme,132) and RAKh is expressed in rat liver and kidney. This also diŠers from the ubiquitous tissue distribution of AKR1C3. (3) Mouse AKR1C14133) and rat AKR1C9134) are 3a-HSDs, exhibiting high reductase activities toward various nonsteroidal carbonyl compounds. In this respect, the rodent enzymes are counterparts of human AKR1C4, but they do not exhibit 20a- or 17b-HSD activity that is detected at low levels for the human enzyme.86) The rodent enzymes exhibit a ubiquitous tissue distribution, which is also diŠerent from the liverspeciˆc expression of human AKR1C4. (4) There has been no report on a mouse or rat NADP(H)-dependent 3a-HSD that has similar properties to human AKR1C2. Recent genomic analyses have shown that eight and nine genes for enzymes belonging to the AKR1C subfamily are present in the mouse and rat genomes, respectively. The proteins include the above rodent HSDs and partially characterized mouse aldo-keto reductases

10

Toshiyuki MATSUNAGA et al. Table 5. AKR1C enzymes speciˆcally expressed in mouse and rat tissues. The genes exist as a cluster on chromosomes 13 and 17 for the genomes of mouse and rat, respectively. Mouse AKR1C enzymes

Gene (product)a) Akr1c12 (AKR1C12) Akr1c13 (AKR1C13) Akr1c19 (AKR1C19) Akr1c21 (AKR1C21) Akr1c20 (AKR1C20)

Characteristics NAD(H)-preferring 17b-HSD with moderate DD activity and low 3(20)a-HSD activity. NAD(H)-preferring DD, oxidizing alicyclic alcohols, cis-and trans-dihydrodiols, and nerol. It reduces dicarbonyl compounds and some aromatic aldehydes. NADH-preferring reductase speciˆc for dicarbonyl compounds. It slowly oxidizes 3-hydroxyhexobarbital. NADP(H)-preferring 3(17)a-HSD with DD activity towards cis-dihydrodiols. It reduces dicarbonyl compounds, and some aromatic aldehydes and ketones. NADPH-preferring reductase for dicarbonyl compounds with aromatic ring(s). It exhibits low 3a(17b)-HSD activity.

Rat AKR1C enzymes Gene (product)a)

Characteristics

LOC36773 (AKR1C24) Akr1c12predicted (AKR1C16)

Speciˆcity for cofactors and substrates is essentially the same as that of AKR1C12. Speciˆcity for cofactors and substrates is essentially the same as that of AKR1C13. It slowly oxidizes some 3a-, 17b- and 20a-hydroxysteroids.

LOC307096 (RAKi)

NADH-preferring reductase for dicarbonyl compounds.

Akr1c21predicted (RAKg)

Speciˆcity for cofactors and substrates is essentially the same as that of AKR1C21.

LOC498790 (AKR1C17)

NAD(H)-preferring 3a-HSD for bile aids, 5b-pregnanes, and 4-androstenes. It reduces dicarbonyl compounds and some aromatic aldehydes. NADPH-preferring reductase with broad speciˆcity for various aliphatic and aromatic carbonyl compounds, quinones, and 17-ketoandrostanes.

Akr1c11predicted (AKR1C15) a)

Products are named according to the nomenclature of the AKR superfamily. RAKi and RAKg are tentative names, because they are not assigned in this superfamily.

(AKR1C12 and AKR1C13),135,136) but the functions of the other enzymes have not been studied. We have isolated the cDNAs for AKR1C12, AKR1C13 and other functionally unknown enzymes from mouse and rat tissues, and have characterized the recombinant enzymes. Table 5 summarizes the genes for the enzymes and their characteristics, which include our unpublished results. The tissue distributions of the enzymes, and their relationship with previously isolated enzymes and proteins are presented below. (1) Mouse AKR1C12 has the same sequence as AKRa, which is encoded by the interleukin-3-regulated gene.136) The rat counterpart exhibiting high sequence identity (90z) is AKR1C24, one form (TBER1) of the two reductases for 6-tert-butyl-2,3-epoxy-5-cyclohexene1,4-dione,137) and may be identical to RAKf, which was previously isolated as a partial cDNA.138) The recombinant AKR1C12 and AKR1C24 utilize NAD(H) as a preferred cofactor, and e‹ciently oxidize various 17bhydroxysteroids. In addition, they exhibit moderate DD activity, and low 3a- and 20a-HSD activities, as well as high reductase activity toward a-dicarbonyl compounds and aromatic aldehydes. As AKR1C12 was found as a stomach aldo-keto reductase,135) both AKR1C12131) and AKR1C24 are highly expressed in the gastrointestinal tract and liver, and at low levels in other tissues of the two animals. (2) Mouse AKR1C13 is probably the same enzyme as NAD＋-preferring DD, which is designated as AKR1C22 in the AKR superfamily,139) because they diŠer only by four amino acids. Its amino acid sequence is also highly similar to that of mouse morphine 6dehydrogenase (EC 1.1.1.218),140) suggesting the identi-

ty of the two enzymes. The rat counterpart (92z sequence identity) is AKR1C16, which is encoded in the cDNA for RAKb.138) The properties of the recombinant AKR1C16 are essentially identical to those of mouse AKR1C13, except that the rat enzyme shows low dehydrogenase activity toward some 3a-, 17b- and 20ahydroxysteroids. The tissue expression patterns of the mRNAs for AKR1C13131) and AKR1C16 are similar to those of AKR1C12 and AKR1C24. In rat liver, the activity of AKR1C16 is lower than that of AKR1C24. Thus, the physiological roles of AKR1C13 and AKR1C16 remain unknown, although the enzymes may be involved in the oxidative metabolism of morphine. (3) Mouse AKR1C19 shows high NADH-linked reductase activity only toward dicarbonyl compounds, but its NAD＋-linked dehydrogenase activity is low and directed toward some alcohol substrates.141) The enzyme is thought to act as a reductase based on the low inhibition of its reductase activity by NAD(P)＋. The rat counterpart is RAKi (91z sequence identity), and the recombinant RAKi shows similar NADH-linked reductase activity and tissue distribution to AKR1C19.131) (4) Mouse AKR1C21 is a NADP(H)dependent 3(17)a-HSD that is identical to DD puriˆed from kidney.142–144) Although the enzyme is highly expressed in the kidney, its mRNA is detected in other tissues. The rat counterpart of AKR1C21 is RAKg, the sequence identity being 86z. NADP＋-linked 3(17)a-HSD activity was also observed for recombinant RAKg, but the mRNA for RAKg is only expressed in liver and kidney, in which its alternative splicing form is detected. (5) AKR1C17 and AKR1C15 are rat-speciˆc

Mammalian Carbonyl-Reducing Enzymes

enzymes. AKR1C17 has been identiˆed as a kidneyspeciˆc 3a-HSD that diŠers from ubiquitous NADP(H)dependent 3a-HSD (AKR1C9) with respect to its cofactor preference for NAD(H) compared to NADP(H), and substrate speciˆcity for bile acids, 5b-pregnanes and 5a-androstanes. AKR1C15 is a NADPH-dependent reductase, which is highly expressed in rat lung. It reduces a variety of carbonyl compounds with low Km values (0.3–20 mM). In particular, the ability of the enzyme to reduce aliphatic ketones and aldehydes is a unique characteristic that is not observed for the other human and rodent enzymes in the AKR7A and AKR1C subfamilies. The broad substrate speciˆcity of the enzyme resembles that of mouse lung CBR2, and the only diŠerence being in the steroid speciˆcity: 5 bAKR1C15 reduces the 17-keto groups of 5a W androstanes, whereas mouse CBR2 reduces 3ketosteroids. Since the CBR2 gene has not been identiˆed in the rat genome and such a tetrameric CBR is indeed absent in rat lung, AKR1C15 may play an important role in the pulmonary metabolism of carbonyl compounds derived through lipid peroxidation, and androgens and xenobiotics. (6) Mouse AKR1C20 shows 89z sequence identity with mouse 17b-HSD type 5 (AKR1C6), and its mRNA is expressed only in the liver.131) The recombinant AKR1C20 exhibits low 17bHSD and moderate 3a-HSD activities using NADP(H) as the preferred cofactors. However, the Vmax W Km values for 3a-hydroxy- and 3-keto-steroids of AKR1C20 (0.01–20 units W mg W mM) are much lower than those of mg W mM). mouse 3a-HSD, AKR1C14 (200–4860 units W AKR1C20 shows high reductase activity towards adicarbonyl compounds with aromatic ring(s), but does not reduce aromatic and aliphatic ketones. The genes for all the enzymes belonging to the AKR1C subfamily exist as a cluster on chromosomes 10, 13 and 17 for the genomes of humans, mouse and rat, respectively. This genomic organization suggests that these genes arose from ancient duplication events followed by divergence. As shown in a phylogenetic analysis of the results of the multiple sequence alignment (Fig. 1), the enzymes form a monophyletic gene family comprised of three separate clades: the rodent 3(17)a-HSD (AKR1C21 and RAKg) clade, the rodent 3a-HSDs and 20a-HSD clade, and the clade of other enzymes. The phylogenetic tree reveals that initial duplication of the ancestral gene common to the AKR1C enzymes shared among the these clades, and the ancestral gene was subsequently duplicated also, giving rise to three clusters: the human enzymes, rodent enzymes (AKR1C15, RAKh, AKR1C6 and AKR1C20) and other rodent enzymes (AKR1C12–RAKi). As described above, there are signiˆcant diŠerences in the multiplicity and tissue distribution of the enzymes belonging to the AKR1C subfamily between humans

11

Fig. 1. Phylogenetic relationship among the human, rat and mouse members of the AKR1C subfamily. Cluster analysis of the human (Hs), rat (Rn) and mouse (Mm) enzymes were performed using Clustal W145) and the tree was drawn with the TreeView program.146)

and laboratory animals. Although the substrate speciˆcities for ketone-containing drugs of all the enzymes have not been studied comparatively, such a diŠerence between the human and rodent enzymes may be responsible for the species diŠerence in drug metabolism. In addition, the occurrence of many HSDs belonging to the AKR1C subfamily in mouse and rat tissues suggests that the enzyme system to regulate cellular concentrations of active steroids in laboratory animals is more complex than that in humans. Human AKR1C1AKR1C4 are potential targets for the treatment of several cancers, endometriosis and anxiety, and selective inhibitors of the individual enzymes have been suggested to be new pharmaceutical agents for the treatment of these diseases.86) The multiplicity of the mouse and rat enzymes should be considered when the eŠects of inhibitors are examined using laboratory animals. Conclusion Recent genomic and cDNA analyses suggested that the reductive metabolism of xenobiotic carbonyl compounds is mediated by many enzymes that diŠer in intracellular localization. The number of the human enzymes is 12, which include uncharacterized CBR4. Most enzymes function in the cellular metabolism of

12

Toshiyuki MATSUNAGA et al.

their natural components, and convert toxic and lipidsoluble carbonyl compounds into less-reactive alcohols because of their broad substrate speciˆcity. Thus, the omnipresent reductive potential must play an important part not only in normal cellular physiology, but also in the metabolism of ketone-containing drugs and xenobiotic carbonyl compounds ingested. The literature indicates that human CBR1, HSD11B1, AKR1C1, AKR1C2 and AKR1C4 are major reductases of ketonecontaining drugs, but suggests that their speciˆcity and catalytic e‹ciency for several therapeutic drugs are signiˆcantly diŠerent. In future studies on the metabolic fate of ketone-containing drugs, it will be necessary to comparatively examine the substrate speciˆcities of all the enzymes, expecting roles of the newly-found enzymes, CBR3, CBR4, DHRS4 and L-xylulose reductase, in the metabolism. In rats and mice, additional ˆve or six reductases belonging to the AKR1C subfamily are suggested to be involved in the metabolism of xenobiotic carbonyl compounds. In addition, the tissue distributions of rodent 20a-HSDs, 17b-HSD type 5 isozymes and 3aHSDs are signiˆcantly diŠerent from those of the corresponding human enzymes, AKR1C1, AKR1C3 and AKR1C4, which play important roles in pre-receptor regulation of steroid actions. Such additional enzymes are perhaps involved in the animal-speciˆc cellular metabolism. This, together with the diŠerences in the tissue distributions of the other HSDs, may be responsible for the diŠerence in the metabolism of ketone-containing drugs between humans and laboratory animals.

8)

9)

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12)

13)

14)

References 1)

2) 3)

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