Human 20α–Hydroxysteroid Dehydrogenase: Crystallographic and Site-directed Mutagenesis Studies Lead to the Identification of an Alternative Binding Site for C21-steroids

Couture, Jean-François; Legrand, Pierre; Cantin, Line; Luu‐The, Van; Labrie, Fernand; Breton, R.

doi:10.1016/s0022-2836(03)00762-9

Cited by 73 publications

(96 citation statements)

References 47 publications

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“…By contrast, Diol is an inactive androgen with a low affinity for the androgen receptor (K d = 10 −6 M). Therefore, the consequence of the reaction catalyzed by AKR1C2 is the elimination of the androgen signal in the target tissue.The crystal structures of several AKR1Cs including those of AKR1C2 have now been solved (13)(14)(15)(16)(17). A number of common features have been noted including the overall structure, the cofactor binding site and the active site.…”

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confidence: 99%

Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

Jin

Penning

2006

Biochemistry

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Human type 3 3α-Hydroxysteroid dehydrogenase, or Aldo-Keto Reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5α-dihydrotesterone (DHT, potent androgen) to form 3α-andostanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k cat for the NADPH-dependent reduction of DHT catalyzed AKR1C2 is 0.033 s −1 . We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k cat value. Stopped-flow experiments to measure the formation of the AKR1C2·NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on k cat (1.5) and a slightly larger effect on k cat /K m (2.1) were observed in the steady state. In the transient state, the maximum rate constant for single turnover of DHT (k trans ) was determined to be 0.11 s −1 for the NADPH-dependent reaction, which was about 4 fold greater than the corresponding k cat . k trans was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent D k trans of 3.5. Thus the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the 3-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s −1 ), the release of the steroid product (0.081 s −1 ), and the release of the cofactor product (0.21 s −1 ), combine to yield the overall observed low turnover number.Cytosolic hydroxysteroid dehydrogenases (HSDs), which catalyze the NADPH dependent reduction of ketosteroids to hydroxysteroids, are members of the aldo-keto reductase (AKR) superfamily 1C subfamily (1-3). By interconverting potent steroid hormones with their cognate inactive metabolites, AKR1C enzymes can regulate the occupancy of hormone receptors in Address all correspondence to: Dr. Trevor M. Penning, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, Phone: 215-898-9445, FAX: 215-573-2236; Email: penning@pharm.med.upenn.edu. 1 Abbreviations and trivial names: HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; AKR1C2, human type 3 3α-HSD/ bile acid binding protein; DHT, 5α-dihydrotestosterone or 17β-hydroxy-5α-androstan-3-one; Diol, 3α-androstanediol or 5α-androstane-3α,17β-diol; AKR1C9, rat liver 3α-HSD; AKR1B1, human aldose reductase; AKR2B5, yeast xylose reductase; KIE, kinetic isotope effect; 5R2, type 2 5α-reductase. 2 The nomenclature for the aldo-keto reductase superfamily was recommended by the 8 th International Symposium...

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Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

Jin

Penning

2006

Biochemistry

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“…Mouse 3(17)aHSDs share high amino acid sequence identity (69-75%) with rat 3a-HSD (AKR1C9), human 3(20)a-HSD (AKR1C1) and human 3a-HSDs (AKR1C2, AKR1C3 and AKR1C4) and rabbit 3a/17b/20a-HSD (AKR1C5). Recent crystallographic and site-directed mutagenesis studies of AKR1C1, 28) ) of the mouse enzymes differs from those of the other HSDs, and may also be related to the substrate specificity, because the importance of the C-terminal residues for the discrimination of the incoming steroid substrates has been suggested by crystallographic and site-directed mutagenesis studies of AKR1C5, 31) AKR1C1 28,32) and AKR1C4.…”

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Characterization of Two Isoforms of Mouse 3(17).ALPHA.-Hydroxysteroid Dehydrogenases of the Aldo-Keto Reductase Family

Ishikura

Usami

Nakajima

et al. 2004

Biological & Pharmaceutical Bulletin

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Mouse kidney contains two 3(17)a a-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two 3(17)a a-HSDs from a total RNA sample of mouse kidney by reverse transcription-PCR. The identity of the cDNAs was confirmed by characterization of the recombinant enzymes that showed the same molecular weights, pI values, pH optima, substrate specificity and inhibitor sensitivity as those of the enzymes from mouse kidney. We also found that the recombinant enzymes reduce precursors of neuroactive progesterone derivatives, 5a a-dihydrotestoserone, deoxycorticosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and estrone at low K m values of 0.3-2 m mM. The two enzymes belonged to the aldo-keto reductase (AKR) family, and their 323-amino acid sequences differed only by five amino acids. The sequences of the two isoforms are identical to those of proteins that are predicted to be encoded in a gene for AKR1C21 in the database of the mouse genome. However, the mRNAs for the two isoforms were expressed in mouse kidney and other tissues, in which their expression levels were different. The results indicate an important role of 3(17)a a-HSD in controlling the concentrations of various steroid hormones in the mouse tissues, and suggest the existence of two genes for the two isoforms of the enzyme.

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“…Crystallographic studies of mammalian HSDs in the AKR1C subfamily 11,[15][16][17][18][19][20][21] show that the enzymes adopt a (β/α) 8 triose-phosphate isomerase (TIM)-barrel structure characterized by a deep hydrophobic active-site located near the Cterminal end of the β-barrel. The coenzyme is situated at the base of the active-site cavity with the carbonyl or hydroxyl group of the substrate interacting with the catalytically important residues, H117 and/or Y55.…”

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confidence: 99%

Probing AKR1C30 and AKR1C31 with Site-Directed Mutagenesis: Identifying the Roles of Residues 54 and 56 in the Binding of Substrates and Inhibitors

Endo

Arai

Matsunaga

et al. 2014

Biological & Pharmaceutical Bulletin

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Five rabbit aldo-keto reductases (AKRs) that participate in the reduction of drug ketones and endogenous ketosteroids have recently been cloned and characterized. Among them, AKR1C30 and AKR1C31 show the highest amino acid sequence identity of 91%, but markedly differ in their substrate specificity and inhibitor sensitivity. AKR1C30 reduces two drugs (ketotifen and naloxone) and 17-keto-5β-androstanes, whereas AKR1C31 does not reduce the two drugs, but is active towards loxoprofen and various 3/17/20-ketosteroids. In addition, AKR1C30 is selectively inhibited by carbenoxolone, valproic acid and phenobarbital. Residues A54 and R56 are located adjacent to the catalytic residue Y55 of AKR1C30. To clarify the determinants for the substrate specificity and inhibitor sensitivity of AKR1C30, we performed the mutagenesis of A54 and R56 to the corresponding residues (L and Q) of AKR1C31. The A54L mutation produced an enzyme that had almost the same substrate specificity as AKR1C31 and decreased the sensitivity to the inhibitors except for carbenoxolone. The R56Q mutation decreased the affinity for only carbenoxolone among the substrates and inhibitors. Thus, the difference in the properties between the two enzymes can be attributed to their residue difference at positions 54 and 56.Key words aldo-keto reductase; carbenoxolone; drug ketone reductase; hydroxysteroid dehydrogenase; sitedirected mutagenesis Carbonyl reduction into the alcohol metabolites is an important Phase-I metabolism of carbonyl-containing drugs. 1,2)To detoxify and excrete the chemically divergent carbonyl compounds, nicotinamide adenine dinucleotide phosphate reduced form (NADPH)-dependent reductases with broad substrate specificity for carbonyl compounds exist in multiple forms in the body. The enzymes with known primary structures are grouped into two distinct protein families: the shortchain dehydrogenase/reductase 1,3) and aldo-keto reductase (AKR) 2-4) superfamilies. Major human enzymes responsible for the reduction of drug ketones in the AKR superfamily are four cytosolic hydroxysteroid dehydrogenase (HSD) isoforms, 2,3) which belong to the AKR1C subfamily and named AKR1C1, AKR1C2, AKR1C3 and AKR1C4.In laboratory animal rats or mice, five NADPH-dependent AKRs similar to the human HSD isoforms were identified and suggested to reduce xenobiotic carbonyl compounds. 3,[5][6][7][8][9][10] In rabbits, such AKRs are six, which are AKR1C5 acting as 17β/20α-HSD, 11,12) AKR1C29 (that is identical to 3-hydroxyhexobarbital dehydrogenase and exhibits 3α/3β/17β/20α-HSD activity), 13) AKR1C30 (that is identical to naloxone reductase type 1 and acts as 17β-HSD), AKR1C31 (that acts as 3α/17β/20α-HSD), AKR1C32 (that is identical to loxoprofen reductase and acts as 3α/20α-HSD) and AKR1C33 (that is identical to naloxone reductase type 2 and mainly acts as 3α-HSD).14) The rabbit AKRs share more than 73% amino acid sequence identity, which is 94% between AKR1C30 and AKR1C31. Despite of the high sequence identity, AKR1C30 and AKR1C31 differ in their specificity for dr...

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Human 20α–Hydroxysteroid Dehydrogenase: Crystallographic and Site-directed Mutagenesis Studies Lead to the Identification of an Alternative Binding Site for C21-steroids

Cited by 73 publications

References 47 publications

Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

Multiple Steps Determine the Overall Rate of the Reduction of 5α-Dihydrotestosterone Catalyzed by Human Type 3 3α-Hydroxysteroid Dehydrogenase: Implications for the Elimination of Androgens

Characterization of Two Isoforms of Mouse 3(17).ALPHA.-Hydroxysteroid Dehydrogenases of the Aldo-Keto Reductase Family

Probing AKR1C30 and AKR1C31 with Site-Directed Mutagenesis: Identifying the Roles of Residues 54 and 56 in the Binding of Substrates and Inhibitors

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