NADPH: 3-hydroxysteroid

This enzyme [EC 1.1.1.146] catalyzes the reaction of an 11/3-hydroxysteroid with NADP+ to produce an 11-oxosteroid and NADPH. 

Hydroxysteroid dehydrogenase (NAD ) [EC 1.1.1.150] catalyzes the reaction of pregnan-21-ol with NAD to produce pregnan-21-al and NADH. Other 21-hydroxy corticosteroids can also serve as substrates. 21-Hydroxysteroid dehydrogenase (NADP ) [EC 1.1.1.151] catalyzes the reaction of pregnan-21-ol with NADP to produce pregnan-21-al and NADPH. Other 21-hydroxy-corticosteroids can also serve as substrates. 

Reactions catalyzed by 11 (3-hydroxysteroid and 17(3-hydroxysteroid dehydrogenases, (a) 11 (3-hydroxysteroid dehydrogenase type 1, an NADPH-dependent enzyme, catalyzes the conversion of the inactive steroid, cortisone, to cortisol, which is the biologically active glucocorticoid. 11 (3-hydroxysteroid dehydrogenase type 2, an NAD+-dependent enzyme, catalyzes the reverse direction, (b) 17(3-hydroxysteroid dehy-drogenase type 1, an NADPH-dependent enzyme, catalyzes the reduction of estrone to estradiol. Type 2, an NAD+-dependent enzyme, catalyzes the oxidation of estradiol to estrone. Type 3, an NADPH-dependent enzyme, catalyzes the reduction of androstene dione to testosterone. Type 4, an NAD+-dependent enzyme, catalyzes the oxidation of estradiol to estrone, and androstenediol to dehydroepiandrosterone. 

Amino acids important in cofactor and catalysis in human 1 lb-hydroxysteroid dehydrogenase types 1 and 2. (a) 1 lb-HSD type 1. Preference of 1 lb-HSD type 1 for NADPH resides in lysine-44 and arginine-66, which have positively charged side chains that stabilize the binding of the 2 -phosphate on NADPH. These residues also counteract the repulsive interaction between glutamic acid 69 and the phosphate group,... 

This interconversion is catalaysed by 17)3-hydroxysteroid dehydrogenase (17/3-HSD), an enzyme generally found in the ER of numerous tissues such as adrenal, liver, testis, ovary and kidney. Like many of the enzymes described above, there appear to be different forms [52,87]. For example, rat adrenal cytosol and ER contain separate 17/3-HSDs, with NADH as the preferred cofactor. The rat testicular enzyme, however, prefers NADPH. Guinea-pig liver also contains two 17j3-HSDs, one solubilized from cytosol, the other associated with the ER [88], These enzymes exhibit different activities towards C19 steroids, the cytosolic one preferring 5/3-reduced 17-oxosteroids and the microsomal counterpart being involved with 5a-reduced steroids, such as 5a-DHT. In this case, the product of the reaction would be 5a-an-drostane-3,17-dione. 

NADPH Glucose-6-phosphate -> 6-phosphogluconic addf Acetyl CoA —> palmitate Steroids 2 hydroxysteroids Glucose-6-phosphate dehydrogenase Fatty add synthetase Cytochrome P-450 system... 

Rahier and his co-workers also characterized the activities of a sterol C-4 methyl oxidase (SMO), a 4-carboxysterol-3-hydroxysteroid dehydrogenase/ C-4 decarboxylase (3-HSD/D) and an NADPH-dependent 3-oxosteroid reductase in order to define the steps involved in C-4 demethylation in plants (Pascal et al, 1993 Rondet et al, 1999). Only recently, they have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3-HSD/D. Transformation of a yeast ergosterol auxotroph mutant, which lacks 3-HSD/D activity, with either of these cDNAs restored ergosterol biosynthesis in the yeast mutant (Rahier et al, 2006). 

NADPH 3p-hydroxysteroid 5fS-oxidoreductase (3p-HS-5p OR) The 3p-HS-5p-OR catalyses the conversion of 5 3-pregnane-3,20-dione to 5p-pregnane-3p-ol-20-one. It was found to be a soluble protein (Gartner and Seitz, 1993). The reverse reaction was observed, yielding 5 3-pregnane-3,20-dione when using 5(3-pregnane-3(3-ol,20-one and NADP as a substrate and co-substrate, respectively. 

B. Del Bello, E. Maellaro, L. Sugherini, A. Santucci, M. Comporti and A.F. Casini, Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3a-hydroxysteroid dehydrogenase, Biochem. J. 304 (1994) 385-390. 

A 3a-hydroxysteroid dehydrogenase active on 7a-hydroxy-5 -cholestan-3-one and 7a,12a-dihydroxy-5 -cholestan-3-one (cf. Fig. 3), was partially purified (about 300-fold) from rat Uver cytosol by Berseus [112,113]. NADPH was required as cofactor and hardly any activity was observed with NADH. The preparation was also active towards 3-oxo steroids of the Cjg, C21 and C24 series, and in these cases appreciable activity was obtained also with NADH. The mechanism of reduction involves a stereospecific transfer of a hydride ion from the 4A position of NADPH to the 3)S position of the steroid [114],... 

The stereochemistry of the reduction was elucidated by use of [4A- H]NADPH, [4B- H]NADPH or the comparable forms of NADH only the tritium from the [4A- H]NADPH or [4A- H]NADH was incorporated into the product, probably in the 3 position [170]. Whether more than one 3-hydroxysteroid dehydrogenase active on 3-oxo-bile acids exists in cytosol of liver cells remains to be determined. 

Microsomal fractions contain 3a- and 3 -hydroxysteroid dehydrogenases which do not appear to be derived from cytosol [171]. Thus, microsomal 3a-hydroxysteroid dehydrogenase preferentially utilized [4B- H]NADH instead of [4A- H]NADPH preferred by the cytosolic enzyme. Microsomal 3)3-hydroxysteroid dehydrogenase was inactive toward ethanol, and thus is not an active component of alcohol dehydrogenase [172]. These dehydrogenases were active with ,9, C21, C24 and C27... 

Although cholesterol is the major source of 5)9-bile acids, an unsaturated acid, 3)8-hydroxy-5-cholenic acid [174] has been found in meconium, mainly as the sulfate [175], in bile of a boy with a deficiency of 3)8-hydroxysteroid dehydrogenase [176], and in urine of healthy persons and individuals with liver disease [164]. The details of metabolism of 3)8-hydroxy-5-cholenic acid to lithocholate have not been entirely elucidated, but the mechanism for conversion of the 3/8-hydroxy-A to the 3-oxo-A derivative has been formulated in the C27 series (cf. Chapter 9). Briefly, the 3)8-ol is dehydrogenated by a microsomal enzyme fortified with NAD to provide the 3-oxo-A system [177,178]. Whether a A - A" isomerase is essential is not known, since there is no direct evidence for the formation of the intermediary 3-oxo-A system the rate-limiting step is the dehydrogenation of the 3)8-ol which may prevent accumulation of the 3-oxo-A system [177]. The reduction of the double bond at 4-5 to the 5)8- or 5a-bile acid is catalyzed by the respective A -3-oxosteroid 5)8- or 5 -reductase obtained from liver cytosol [170], and has been purified about 10-fold [178]. The formation of the 3-oxo-5/9 derivative requires the enzyme and NADPH the proton from the A side (4A-NADPH) appeared in the product as the 5)8-H, whereas the proton at C-4 is derived from the aqueous medium. Formation of the 5a derivative requires (4B-NADPH) in a similar mechanism (Fig. 4) [179], Reduction of the 3-0X0 product is then catalyzed by 3a-hydroxysteroid dehydrogenase as discussed above. 

Besides the involvement in the de novo biosynthesis of BH4, SR may also participate in the pterin salvage pathway by catalyzing the conversion of sepiapterin (Figure 14, 47) into 7,8-dihydrobiopterin (46) that is then transformed to BH4 by dihydrofolate reductase (DHFR EC 1.5.1.3). Both reactions consume NADPH. Although SR is sufficient to complete the BH4 biosynthesis, a family of alternative NADPH-dependent aldo—keto reductases, including carbonyl reductases (CR), aldose reductases (AR), and the 3a-hydroxysteroid dehydrogenase type 2 (AKR1C3) may participate in the diketo reduction of the carbonyl side chain in Moreover, based on the discover) of the autosomal recessive deficiency for SR, which presents... 

The reduction of a 3-oxosteroid, catalyzed by 3a-hydroxysteroid dehydrogenase from rat liver, has been shown to involve transfer of a hydride ion from the A-side of NADPH to the 3j -position of the steroid (44,52). The oxidation of 4-cholestene-3a,7a-diol into 7a-hydroxy-4-cholesten-3-one, catalyzed by the same enzyme preparation, has also been shown to involve the A-side of NADPH (52). It remains to be established whether or not Zl -3a-hydroxysteroid dehydrogenase activity is due to enzyme(s) different from 3a-hydroxysteroid dehydrogenase(s). 

In the next step, the conversion of 7a-hydroxy-4-cholestene-3-one into 5j8-cholestene-3a, 7a, 12a-triol involves the 12 hydroxylation of the substrate by a NADPH microsomal enzyme. The product of the reaction, the 7a, 12a-dihydroxy-4-cholestene-3-one, is converted into the 7a, 12a-dihydroxy-5j8-cholestene-3-one by a soluble A, 3-oxysteroid-5j8-reductase, and 3a-hydroxysteroid dehydrogenase yields the final intermediate 5j -cholestene-3a, 7a, 12a-triol. 

The 3 hydroxysteroid reductase of rat liver which catalyzes the conversion of XXX to cholestanol (XXXI) was localized also in the microsomal fractions, and shown to provide the epimeric alcohols in a ratio of 10 1 (3/3 3a) in the presence of NADPH. The enzyme was not inhibited by cholestanol, but pronounced inhibition was noted with 7-keto- or 7a-hydroxycholestanoI (XXXII) or zl -cholestenone (XXVIII) (120). This enzyme differs from the Cj9 steroid reductase, since the latter utilize NADH equally well and provides predominantly the 3a-ol. 

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