20/3-Hydroxysteroid dehydrogenase

Characteristics of la- and 7)8-hydroxysteroid dehydrogenase (HSDH) from intestinal bacteria  

The epimerizalion of the 7a-hydroxyl group can occur either by intra- or interspecies mechanisms [16]. However, it is difficult to quantitatively assess the degree of 7-hydroxy epimerization in vivo because this transformation competes with the irreversible 7-dehydroxylation of bile acids (Section VI). 7a-HSDH activity has been reported in several genera of intestinal bacteria however, the most complete characterization of this enzyme has been carried out with the enzyme isolated from Escherichia coli [37] and Bacteroides sp. [29,38,39] (Table 2). Both enzymes used both free and conjugated bile acids as substrates, showed alkaline pH optima and lower values for dihydroxy than for trihydroxy bile acids. However, cell extracts prepared from Bacteriodes sp. contained both NAD- and NADP-depen-dent 7 -HSDH activities whereas, extracts from E. coli contained only an NAD-de-pendent enzyme activity. Additional studies showed that the two 7a-HSDH activities detected in Bacteriodes sp. differed in molecular weight, differential heat inactivation and Mn requirement, suggesting the presence of two distinct enzymes [29]. 

The NAD-dependent 7a-HSDH was found to be associated with both cytoplasmic and cell membrane fractions during the preparation of spheroplasts of B. fragilis [20]. Moreover, the activity of this enzyme in B. fragilis increased 3-5-fold as the cultures entered stationary growth phase. However, the mechanism of regulation of enzyme synthesis is not yet known. 

The 7)8 epimers of cholic and chenodeoxycholic acid are frequently detected in feces and bile of man. However, the epimerization of the 7)8-hydroxy bile acids by 

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Testosterone, the principal male sex steroid hormone, is synthesized in five steps from cholesterol, as shown below. In the last step, five isozymes catalyze the 17/3-hydroxysteroid dehydrogenase reactions that interconvert 4-androstenedione and testosterone. Defects in the synthesis or action of testosterone can impair the development of the male phenotype during embryogenesis and cause the disorders of human sexuality termed male pseudohermaphroditism. Specifically, mutations in isozyme 3 of the 17/3-hydroxysteroid dehydrogenase in the fetal testis impair the for-... 

Like all steroids, aldosterone enters the target cell and combines with cytosolic mineralocorticoid receptor. Such receptors are not entirely specific for aldosterone and will also bind cortisol, the principal glucocorticoid hormone. The receptors are protected from cortisol activation by 11 3 hydroxysteroid dehydrogenase which... 

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

Table 6 Comparison of the Determination of 11-/3-Hydroxysteroid Dehydrogenase Activity by HPLC, MEKC, and MEEKC...

A. 17,20 lyase is required for androgen synthesis, cyclooxygenase for prostaglandin production, 11- 3-hydroxysteroid dehydrogenase-2 acts as a reductase-converting cortisol to its inactive 11-keto derivative cortisone, whereas 18-hydroxylase is required for aldosterone production. 

Mensah-Niagan AG, Feuilloley M, Dupont E, et al Immunocytochemical localization and biological activity of 3 (3-hydroxysteroid dehydrogenase in the central nervous system of the frog. J Neurosci 14 7306-7318, 1994... 

Fig. 3. Gonadal steroid biosynthetic pathway and the catalytic enzymes 1) cytochrome P-450scc 2) -hydroxysteroid dehydrogenase 3) 17a-hydroxylase (P-450scc17) 4) 17,20-desmolase or 17,20-lyase 5) 17(3-hydroxysteroid dehydrogenase 6) 5a-reductase and 7) P-450 aromatase.

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. 

A similar mechanism has been found for 17(3-hydroxysteroid dehydrogenase (17(3-HSD), the enzyme that regulates the concentrations of estradiol and testosterone in human [5,16,17] (Figure lb). Genetics diseases associated with mutations in this enzyme lead to developmental abnormalities [18]. Enzymes that regulate the concentrations of retinoids [19] and prostaglandins [20] may also have a similar role [6]. 

Alignment of 1 l(3-and 17(3-hydroxysteroid dehydrogenases. As seen in this Figure and Table 1, the sequences of the two 1 l(3-HSDs and four 17(3-HSDs are very divergent. Boxes denote sites where either 5 or 6 residues are conserved, which are... 

Important for the validity of the models that we constructed is the evidence from models of other proteins indicating that two proteins can have as little as 20 to 25% sequence identity and still have very similar 3D structures, especially in a helices and [3 stands [49-52]. Variation is found in the loops and coiled structures. A relevant example for this chapter is the comparison of the tertiary structure of rat dihydropteridine reductase [33] and Streptomyces hydrogenans 20(3-hydroxysteroid dehydrogenase [34]. As noted by Varughese et al. [33], despite less than 20% sequence identity between dihydropteridine reductase and... 

The a helix F in 17J3-HSD-1 [16], 17J3-HSD-2 [17], 17(3-HSD-3 [18], and porcine 17(3-HSD-4 [26] was constructed by modeling on a helix F in 20(3-hydroxysteroid dehydrogenase. Comparisons with other 3D structures [33-37] have demonstrated that this a helix is highly conserved. The modeled dimers were not minimized as a dimer complex to avoid the artifactual adjustment of a helix F side chains. 

Structure of a helix F interface of human 11 (3-hydroxysteroid dehydrogenase types 1 and 2. The a helix F part of the dimer interface on 1 ip-HSD-1 and -2 is shown along with side chains of the highly conserved tyrosine and lysine residues and other residues that are oriented into the cavity that binds substrate and nucleotide cofactor. 

A NADPHBinding Site on 1/(3 -Hydroxysteroid Dehydrogenase Types 1 and2... 

Figure 6a shows the modeled oc helix F interface in human 17 3-hydroxysteroid dehydrogenase type 1 in which phenylalanine-160 and alanine-161 form an anchor. Both residues have important stabilizing interactions across the dimer interface. Alanine-161 is 4.1 A from alanine-161 on the other subunit. Alanine-161 has a hydrophobic interaction with alanine-157, which is in the segment between the conserved tyrosine-155 and lysine-159. There is a hydrophobic... 

Peltoketo H, Isomaa V, Vihko R. Genomic organization and DNA sequences of human 17(3-hydroxysteroid dehydrogenase genes and flanking regions. Eur J Biochem 209 1992 459-466. 

Krozowski Z. 11 (3-hydroxysteroid dehydrogenase and the short chain alcohol dehydrogenase (SCAD) superfamily. Mol Cell Endocrinol 84 1992 C25-C31. 

Baker ME. Protochlorophyllide reductase is homologous to human carbonyl reductase and pig 20(3-hydroxysteroid dehydrogenase. Biochem J 300 1994 605-607. 

Fig. 5.1.2 Cholesterol biosynthesis branch of the isoprenoid biosynthetic pathway. Enzymes are numbered as follows 1 squalene synthase 2 squalene epoxidase 3 2,3-oxidosqua-lene sterol cyclase 4 sterol A24-reductase (desmosterolosis) 5 sterol C-14 demethylase 6 sterol A14-reductase (hydrops-ectopic calcification-moth-eaten, HEM, dysplasia) 7 sterol C-4 demethylase complex (including a 3/ -hydroxysteroid dehydrogenase defective in congenital hemidyspla-sia with ichthyosiform nevus and limb defects, CHILD, syndrome) 8 sterol A8-A7 isomerase (Conradi-Hunermann syndrome CDPX2) 9 sterol A5-desaturase (lathosterolosis) 10 sterol A7-reductase (Smith-Lemli-Opitz syndrome). Enzyme deficiencies are indicated by solid bars across the arrows...

Autosomal recessive mutations in the 17/3-hydroxysteroid dehydrogenase (17/3HSD) 3 gene impair the formation of testosterone in the fetal testis and give rise to genetic males with female external genitalia. Such individuals are usually raised as females, but virilize at the time of expected puberty as the result of increases in serum testosterone. More than 14 mutations have now been identified [20]. 

Bongiovanni AM (1962) The adrenogenital syndrome with deficiency of 3/ -hydroxysteroid dehydrogenase. J Clin Invest 41 2086-2092... 

Rosenfield RL, Rich BH, Wolfsdorf JI, Cassorla F, Parks JS, Bongiovanni AM, Wu CH, Shack-leton CH (1980) Pubertal presentation of congenital A5-3/ -hydroxysteroid dehydrogenase deficiency. J Clin Endocrinol Metab 51 345-353... 

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

HFAA, heptafluorobutyric acid anyhydride BSTFA, N,O-bis(trimethylsilyl)tnfluoroacetamide 3 -hydroxysteroid dehydrogenase BSA, N,O-bis(trimethylsilyl)acetamide MSTFA, N-methyl-N-(trimethylsily)trifluoroacetamide TMS, trimethylsilane SEA, supercritical-fluid extraction PFBCMO, pentafluorobenzylcarboxymethoxime. 

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