Regular ArticleAldose Reductase Is a Major Reductase for Isocaproaldehyde, a Product of Side-Chain Cleavage of Cholesterol, in Human and Animal Adrenal Glands
Abstract
Isocaproaldehyde (4-methylpentanal) is a product of the side-chain cleavage of cholesterol, the first step of steroid biosynthesis. Here, we report the characterization of enzymes responsible for the oxidoreduction of isocaproaldehyde in human, monkey, dog, and rabbit adrenal glands. NADPH-linked isocaproaldehyde reductase activity in the adrenal extracts of the four species was much higher than the NADH-linked reductase and NAD(P)+-linked dehydrogenase activities and was potently inhibited by aldose reductase inhibitors. The major species of isocaproaldehyde reductase purified from the four mammalian adrenal glands were biochemically identical with aldose reductase, and exhibitedKmvalues of 1 μM. The contents of aldose reductase in adrenal glands of the four mammals were relatively high, and its localization in canine adrenal cortex was immunohistochemically demonstrated. In addition, the purified aldose reductases and recombinant human aldose reductase reduced other alkanals and alkenals at lowKmvalues of 2–61 μM, and their catalytic efficiencies were higher than that of human aldehyde reductase. Thus, aldose reductase acts not only as a major reductase for isocaproaldehyde formed from steroidogenesis but also as a scavenger of aldehydes derived from lipid peroxidation in mammalian adrenal glands.
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Porcine aldo-keto reductase 1C subfamily members AKR1C1 and AKR1C4: Substrate specificity, inhibitor sensitivity and activators
2022, Journal of Steroid Biochemistry and Molecular BiologyMost members of the aldo-keto reductase (AKR) 1 C subfamily are hydroxysteroid dehydrogenases (HSDs). Similarly to humans, four genes for AKR1C proteins (AKR1C1-AKR1C4) have been identified in the pig, which is a suitable species for biomedical research model of human diseases and optimal organ donor for xenotransplantation. Previous study suggested that, among the porcine AKR1Cs, AKR1C1 and AKR1C4 play important roles in steroid hormone metabolism in the reproductive tissues; however, their biological functions are still unknown. Herein, we report the biochemical properties of the two recombinant enzymes. Kinetic and product analyses of steroid specificity indicated that AKR1C1 is a multi-specific reductase, which acts as 3α-HSD for 3-keto-5β-dihydro-C19/C21-steroids, 3β-HSD for 3-keto-5α-dihydro-C19-steroids including androstenone, 17β-HSD for 17-keto-C19-steroids including estrone, and 20α-HSD for progesterone, showing Km values of 0.5–11 µM. By contrast, AKR1C4 exhibited only 3α-HSD activity for 3-keto groups of 5α/β-dihydro-C19-steroids, 5β-dihydro-C21-steroids and bile acids (Km: 1.0–1.9 µM). AKR1C1 and AKR1C4 also showed broad substrate specificity for nonsteroidal carbonyl compounds including endogenous 4-oxo-2-nonenal, 4-hydroxy-nonenal, acrolein, isocaproaldehyde, farnesal, isatin and methylglyoxal, of which 4-oxo-2-nonenal was reduced with the lowest Km value of 0.9 µM. Moreover, AKR1C1 had the characteristic of reducing aliphatic ketones and all-trans-retinal. The enzymes were inhibited by flavonoids, synthetic estrogens, nonsteroidal anti-inflammatory drugs, triterpenoids and phenolphthalein, whereas only AKR1C4 was activated by bromosulfophthalein. These results suggest that AKR1C1 and AKR1C4 function as 3α/3β/17β/20α-HSD and 3α-HSD, respectively, in metabolism of steroid hormones and a sex pheromone androstenone, both of which also play roles in metabolism of nonsteroidal carbonyl compounds.
Mouse Akr1cl gene product is a prostaglandin D<inf>2</inf> 11-ketoreductase with strict substrate specificity
2019, Archives of Biochemistry and BiophysicsA mouse gene, Akr1cl, encodes a member of the aldo-keto reductase 1C subfamily (AKR1CL), whose function, however, remains unknown. Here, we show that the recombinant AKR1CL is an NADPH-dependent reductase of prostaglandin (PG) D2 (Km 3.2 μM, kcat 5.6 min−1) and oxidizes 9α,11β-PGF2 (Km 30 μM, kcat 3.3 min−1) in the reverse reaction. In contrast, it did not exhibit oxidoreductase activity towards other PGs (E2, A1, B2 and F2α), steroids and nonsteroidal carbonyls and alcohols, which are substrates of other mammalian AKR1C subfamily enzymes. The enzyme activity was inhibited by estradiol, quercetin, benzbromarone, ethacrynic acid and flufenamic acid, of which estradiol was the most potent competitive inhibitor (Ki 3.2 μM). The mRNA for AKR1CL was expressed abundantly in mouse testis, ovary and adrenal gland, and at low levels in the brain, lung, small intestine and prostate. Thus, AKR1CL is the first PGD2 11-ketoreductase with strict substrate specificity in mammals. The site-directed mutagenesis of P85 in AKR1CL to the corresponding residue, W, in other mammalian AKR1C subfamily enzymes resulted in broad substrate specificity for nonsteroidal carbonyls and alcohols, suggesting that P85 plays a critical role in the strict specificity for PGD2 and 9α,11β-PGF2.
Concomitant and discrete expressions of aldose reductase and sorbitol dehydrogenase in the male reproductive tract
2016, Acta HistochemicaThis study aimed at investigating the expression and localization of the polyol pathway enzymes; aldose reductase (AR) and sorbitol dehydrogenase (SDH), in the male reproductive tract of rat. Gene expression analysis showed maximum expression of AR and SDH in the coagulating glands. Western blot analysis showed a coordinated presence of the two enzymes in the coagulating glands, seminal vesicle and epididymis. Immunohistochemistry showed a concordant expression of the two enzymes in the coagulating gland, which goes well with its function of fructose production in rats. A less concordant expression of the two enzymes in the seminal vesicle was also seen. Discrete expression of AR was seen in the Sertoli cells without SDH. Germ cells including sperm in the seminiferous tubules lacked AR, but SDH was present in all stages of developing germ cells including sperm present in the seminiferous tubules. The epithelial layer of epididymis showed the presence of AR, but it was negligible in vas deferens and prostate. SDH was not seen in the epithelial layer of epididymis, vas deferens or prostate. Though sperm in the seminiferous tubules lacked AR, sperm extracted from cauda showed the presence of both AR and SDH. Immunofluorescence localization of AR and SDH on sperm showed the presence of both the enzymes all over sperm. Discrete expression of AR in the Sertoli cells may be linked to detoxification of a number of metabolism by-products. Similarly, the presence of polyol enzymes on sperm in epididymis and beyond may be to tackle toxic metabolites they may encounter during their journey along the male or female reproductive tract.
Characterization of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehydrogenase activity
2012, Archives of Biochemistry and BiophysicsIn this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo–keto reductase (AKR)1 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing Km of 1.3–9.1 μM and kcat of 1.1–7.6 min−1. The stereospecific reduction was also observed in the metabolism of 5α- and 5β-dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.
Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)
2010, Archives of Biochemistry and BiophysicsA recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo–keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low Km values of 0.8–6.1 μM and 3.7–67 μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (Km = 96–390 μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.
Structure of aldehyde reductase in ternary complex with a 5-arylidene-2,4-thiazolidinedione aldose reductase inhibitor
2010, European Journal of Medicinal ChemistryCitation Excerpt :Aldose reductase (ALR2; EC 1.1.1.21), the first enzyme of the polyol pathway responsible for converting glucose into sorbitol [1], has been isolated and purified from cytosols of a number of mammalian tissues including brain, liver, lens, skeletal muscle and adrenal gland [2–7].
The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and [5-(3-carboxymethoxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (CMD), a potent inhibitor of aldose reductase (ALR2), was determined at 1.99 Å resolution. The partially disordered inhibitor formed a tight network of hydrogen bonds with the active site residues (Tyr50 and His113) and coenzyme. Molecular modelling calculations and inhibitory activity measurements of CMD and [5-(3-hydroxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (HMD) indicated that π-stacking interactions with several conserved active site tryptophan residues and hydrogen-bonding interactions with the non-conserved C-terminal residue Leu300 in ALR2 (Pro301 in ALR1) contributed to inhibitor selectivity. In particular for the potent inhibitor CMD, the rotameric state of the conserved residue Trp219 (Trp220 in ALR1) is important in forming a π-stacking interaction with the inhibitor in ALR2 and contributes to the difference in the binding of the inhibitor to the enzymes.