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Identification des facteurs moléculaires responsables de l'activité particulière de la 17α-hydroxystéroïde déshydrogénase de souris et de la 5β-réductase humaine et étude structurale du domaine de liaison du ligand du récepteur humain des androgènes en complexe avec le EM5744
| Content Provider | Semantic Scholar |
|---|---|
| Author | Faucher, Frédérick |
| Copyright Year | 2008 |
| Abstract | The 5p-reductases (AKR 1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5P-reduction of the C4=C5 double bond found into A4-3ketosteroids, including steroid hormones such as progestérone (Prog) and androstenedione (A4) as well as bile acids precursors 7a-hydroxy-4-cholesten-3-one and 7a,12ot-dihydroxy4-cholesten-3-one. Multiple séquence alignments and mutagenic studies have already identified one o f the residues presumably located at their active site, Glu 1 2 0 , as the major molecular déterminant for the unique activity displayed by 5P-reductases. In order to précise the rôle played by this glutamate in the catalytic activity of thèse enzymes and to elucidate the binding mode and substrate specificity in détail, biochemical and structural studies on human 5p-reductase (h5p-red) have been recently undertaken. The crystal structure of h5P-red in binary complex (NADPH) with that of this enzyme in ternary complex ( N A D P + and 5P-dihydroprogesterone the product of the 5P-reduction o f Prog), has allowed us to détermine the spatial organization of ail residues forming the enzyme active site, including Glu 1 2 0 . W e have found that Glu 1 2 0 does not interacte directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could rather be important for the good positioning of the steroid substrate in the catalytic site. Kinetic characterization of the purified recombinant h5P-red has also revealed that this enzyme exerted a strong activity toward A4 and Prog but was rapidely inhibited by thèse substrates as soon their concentrations reached two-fold their Km value. Crystal structure of the h5P-red in ternary complex with N A D P + and A4 has shown that the quite large steroid-binding site of h5P-red also contained a subsite in which the A4 molécule was found. When bound in this subsite, A4 completely impedes the passage o f another substrate molécule toward the catalytic site. The importance o f this alternative binding site for the inhibition of h5P-red was finally proven by site-directed mutagenesis, demonstrated that the replacement of one of the residues delineating this site (Val309) by a phenylalanine completely abolished the substrate inhibition. Based on our results, we thus propose a realistic scheme for the catalytic mechanism of the C4=C5 double bond réduction catalyzed by h5p-red. 3.4.2 I n t r o d u c t i o n The enzyme 5P-reductase catalyses the réduction of the 4-ene-3-ketosteroids such as progestorone (Prog) and androstenedione (A4), converting them into 5P-dihydro-3ketosteroids steroids. It also transforms the bile acid precursors, 7a-hydroxycholest-4-en-3one and 7a,12oc-dihydroxycholest-4-en-3-one, into their corresponding 5P-reduced derivatives. (Berseus 1967; Berseus and Bjorkhem 1967; Charbonneau and Luu-The 1999) Human 5p-reductase (h5p-red) belongs to the aldo-keto réductase (AKR) superfamily and is the first member o f the 1D subfamily (AKR1D1) which also encompasses the 5preductases from rat (AKR1D2) (Onishi, Noshiro et al. 1991) and rabbit (AKR1D3) . As for the rat enzyme, the h5p-red is highly expressed in the liver (Kondo, Kai et al. 1994) and, at lower levels, in testes and placenta. (Charbonneau and Luu-The 1999; Sheehan, Rice et al. 2005) The h5p-red is well known as playing a major rôle in bile acids synthesis and defects in AKR1D1 gene resuit in high urinary and plasma levels of A4-3-oxo steroids causing liver failures and hemochromatosis which could be lethal in newborn infants. (Clayton, Patel et al. 1988; Setchell, Suchy et al. 1988; Lemonde, Custard et al. 2003) In addition, the h5P-red efficiently transforms Prog into 5p-pregnan-3,20-dione (5P-DHP), an endogenous ligand for the pregnane X receptor and for the constitutive androstane receptor that are implicated in clearance of xenobiotics. (Moore, Parks et al. 2000) This Prog métabolite is also a potent tocolytic steroid (Kubli-Garfias, Medrano-Conde et al. 1979) and its circulating concentration has been found to decrease significantly in association with the active phase o f the first stage of human labour (Sheehan, Rice et al. 2005). Because the 5P-red mRNA-relative abundance in placenta and myometrium also decreased significantly in association with labour (Sheehan, Rice et al. 2005), it has been hypothesized that this enzyme, through the formation of the 5P-reduced metabolites of Prog, could play a rôle in regulating myometrial activity and in onset of spontaneous labour in the human. Actually, AKR1D1 is the only known human enzyme capable to efficiently catalyze the 5P-reduction of A4-3-ketosteroids. Because some 5p-reduced steroids are active molécules and are possibly involved in many physiological functions, such as induction of porphyrin synthesis (Granick and Kappas 1967), increase of red cell and erythroblast formation in the bone marrow (Levere and Granick 1965; Levere, Kappas et al. 1967; Kappas and Granick 1968), and stimulation of the expression of cytochrome P-450dependent drug metabolizing enzyme (Bertilsson, Heidrich et al. 1998), the activity of this enzyme is thus of primary importance. It has previously reported that the h5P-red could be strongly reduced or inhibited by mineralocorticoids, especially by cortisol (Morineau, Marc et al. 1999). The mecanism by which cortisol affects the h5p-red activity is however unknown as well as the capacity of the other steroid hormones to inhibit this enzyme. As the other A K R members, h5p-red is thought to adopt an (a/P)-barrel fold with a cylindrical core of eight parallel P-strands surrounded by eight a-helices running antiparallel to the strands (Borhani, Harter et al. 1992). The carboxy ends of the p-strands are connected to amino ends of the a-helices by loops of varying lengths. Residues forming the active site or participating in substrate binding mainly belong to the loops located at the C-terminal portion of the p-sheet (Rees-Milton, Jia et al. 1998). The cofactor NADPH binds A K R enzymes in an extended conformation with the nicotinamide in the center of the barrel. This cofactor-binding mode contrasts with that found in the 5P~red isolated from leaves of Digitalis plants, the only other 5P-red enzyme whose structure has been determined so far. (Egerer-Sieber, Herl et al. 2006) Indeed, this plant 5P~red contains a dinucleotide-binding Rossmann-fold (Rossmann, Moras et al. 1974) typical of members of the short-chain dehydrogenase/reductase (SDR) superfamily. Although they catalyze the same reaction, it appears that vertebrates and plants 5p-reductases have evolved independently after divergence from the last universal common ancestor. (Gavidia, Tarrio et al. 2007) The réduction reaction catalyzed by the 5P-red would be irréversible Contrarily it is demonstrated that in vitro A K R 1 C members are réversible enzyme since they can perform réduction or oxidisation depending of the cofactor added (NADPH or N A D P + ) . In contrast to A K R 1 C , there is no mention that 5p-reduction is réversible since it is believed that steroid C5 P-face oxidation and the formation of the double bond between C4-C5 is thermodynamically unfavourable. It is important to note that the réduction of a C4=C5 double bond involving proton transfer to the P-face o f the steroid at C5 produces a major structural change with as a resuit that the steroid nucleus goes from a all-trans planar conformation to a cis-trans-trans conformation with the A-ring bent 90° relative to the steroid nucleus (Fig. 1). Enzymes of the A K R 1 D subfamily also differ from the other metabolizing enzymes belonging to the A K R 1 C subfamily by the number of amino acids composing their primary structures and by the identity of residues forming their catalytic tetrad. AKR1C members have ail 323 residues in their séquence while A K R 1 D members have three additional residues (326 residues). Multiple séquences alignment shows that thèse additional residues are ail localized in the N-terminal portion of the protein, around positions (3, 11, and 28). However, the most important différence that distinguishes the two subfamily members is the replacement of the Hisin residue found in the catalytic tetrad of ail AKR1C members (Asp5o, Tyr5 5, Lysg4 and Hisin) by a negatively charged amino acid residue (Glu^o) in the A K R 1 D séquences (Asp53, Tyr^g, Lysg7, and Glui2o). The rôle played by this glutamate residue in the 5(î-reductase activity has been tentatively studied by Jez and Penning (Jez and Penning 1998) who have successfully introduced a stereospecifîc 5p-reductase activity into rat 3oc-HSD simply by mutating to glutamine the conserved Hisin residue within the catalytic tetrad of this enzyme. By studying the effect of the pH on the 5p-reductase activity of their mutated (HisinGlu) 3oc-HSD enzyme, thèse authors have proposed a hypothetical mechanism which implies a direct interaction between two residues of the catalytic tetrad: the Tyr and Glu. According to this mechanism, the protonated glutamate (Glun 7 ) residue established an hydrogen bond with the catalytic tyrosine (Tyr 5 5 ) , thus lowering the pKb of the tyrosine and making it a stronger acid able to promote acid-catalyzed enolization of A4-3-ketosteroids. The enolization of the steroid produces cationic character at C5 which allows the pro-R hydride transfer from NADPH to the p-face of the steroid at C5. This enol intermediate is finally tautomerised to generate the 5P-dihydrosteroid product (Jez and Penning 1998; Penning, Ma et al. 2001). The présence of a glutamate residue in the catalytic tetrad of the A K R enzymes and its close proximity with the catalytic tyrosine thus seems of primary importance for those displaying 5p-reductase activities. However, since no tridimensional structure for an enzyme containing a glutamate in its active site have been determined so far, the exact position of this residue within the cataly |
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| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
