The steroid 15α-hydroxylase ofPenicillium raistrickii I 477 is inducible

Summary It could be shown that the 15-hydroxylase ofPenicillium raistrickii i 477 is inducible by the substrate 13-ethylgon-4-en-3, 17-dione as well as other 3,17-ketosteroids. The influence of inducer concentration, time of this induction and the kind of steroid inducer is described and discussed.     

The suppressive effect of the catatoxic steroid,pregnenolone-16α-carbonitrile,on liver microsomal cholesterol-7α-hydroxylase

The effect of the catatoxic steroid, 3β-hydroxy-20-oxo-5-pregnene-16α-carbonitrile [pregnenolone-16α-carbonitrile (PCN)] on hepatic microsomal cholesterol-7α-hydroxylase, the probable rate-limiting enzyme of bile acid biosynthesis, has been studied. Short term administration (3 days) of PCN in the diet of rats resulted in a significant decrease in the liver microsomal cholesterol-7α-hydroxylase activity, in contrast to a marked stimulation of microsomal cytochrome P-450 and ethylmorphine demethylase activity. PCN significantly depressed the cholesterol-7α-hydroxylase activity in the livers of rats with elevated levels of the enzyme produced by cholestyramine feeding. The results indicate the presence of separate control mechanisms in the regulation of bile acid synthesis and drug metabolism.     

Insight into the orientational versatility of steroid substrates—a docking and molecular dynamics study of a steroid receptor and steroid monooxygenase

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer–Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.     

Synthesis of regioisomeric 17β-N-phenylpyrazolyl steroid derivatives and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

The reaction of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (1) with phenylhydrazine (2a) affords two regioisomers, 17β-(1-phenyl-3-pyrazolyl)androst-3-en-3β-ol (5a) and 17β-(1-phenyl-5-pyrazolyl)androst-5-en-3β-ol (6a). The direction of the ring-closure reactions of 1 with p-substituted phenylhydrazines (2b-e) depends strongly on the electronic features of the substituents. Oppenauer oxidation of 3β-hydroxy-17β-exo-heterocyclic steroids 5a-e and 6a-e yielded the corresponding Δ⁴-3-ketosteroids 9a-e and 10a-e. The inhibitory effects (IC₅₀) of these compounds on rat testicular C_17,20-lyase were investigated by means of an in vitro radioligand incubation technique.     

Cell specific steroid inhibitions in histochemical steroid 3β-ol dehydrogenase activities in man

Summary Histochemical evidence is presented for the occurrence of specific steroid 3-ol dehydrogenase activities in steroidogenic cells of the human ovary, testes and adrenal. The enzymes in the cells of the corpus luteum and adrenal show similar dehydrogenase reactions with some steroid substrates and are inhibited by progesterone, a known physiologic steroid. Theca cells have an activity which is less readily demonstrated and apparently inhibited by both progesterone and DHA. The ovarian hilus cells and the interstitial cells of the testis contain steroid dehydrogenase activity which is inhibited by DHA and not by progesterone. The cellular specificity suggests that the type of activity plays a major role in determining the type of hormone production. The specificity of the steroid inhibitors suggests the possibility of intracellular feedback mechanisms which control the amount of hormone produced.Supported by USPHS Grant No. AMO 3806-07.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Demonstration of a cytochrome P-450-dependent steroid 15β-hydroxylase in Bacillus megaterium

The steroid 15β-hydroxylase system of $\text{Bacillus megaterium}$ was obtained in a cell-free preparation through sonication. The strictly NADPH-dependent 15β-hydroxylase activity, measured using progesterone as substrate, was inhibited by carbon monoxide, SKF 525-A, imidazole and metyrapone, indicating that the reaction is cytochrome P-450-dependent. A 40-fold purification of cytochrome P-450 in cell-free extracts was obtained by chromatography on DEAE-cellulose yielding a concentration of 0.32 nmoles of cytochrome P-450 per mg of protein. This partially purified cytochrome P-450 preparation catalyzed 15β- and 15α-hydroxylation of progesterone in the presence of NaIO₄ or NaClO₂ but not in the presence of NADPH or NADH.     

Scrutiny of electrochemically-driven electrocatalysis of C-19 steroid 1α-hydroxylase (CYP260A1) from Sorangium cellulosum So ce56

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E^0′ of −299 ± 16 mV and −297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.     

Membrane proteins of chromaffin granules. Dopamine β-hydroxylase, a major constituent

1. Soluble lysates and membranes were prepared from chromaffin granules isolated from bovine adrenal medulla. The detergent N-cetylpyridinium chloride was used for solubilizing the membrane proteins, including the membrane-bound dopamine (2,4-dihydroxyphenethylamine) beta-hydroxylase. The solubilized proteins were fractionated by Sephadex chromatography in the presence of N-cetylpyridinium chloride. The major component of the membrane proteins, i.e. chromomembrin A, was identified as the enzyme dopamine beta-hydroxylase. 2. The addition of N-cetylpyridinium chloride to the soluble lysate caused precipitation of up to 96% of the proteins, but only a small proportion of the dopamine beta-hydroxylase activity was precipitated. The only protein demonstrable in the supernatant by polyacrylamide-gel electrophoresis was the protein that has a lower mobility than chromogranin A in disc gel electrophoresis. This component has been identified previously as dopamine beta-hydroxylase. Thus, this method provides an extremely simple isolation procedure for dopamine beta-hydroxylase. 3. A comparison of the membrane-bound and soluble dopamine beta-hydroxylases revealed the identity of these two preparations. Both were activated by N-cetylpyridinium chloride, they migrated identically in polyacrylamide-gel electrophoresis, their amino acid composition was very similar and an immunological cross-reaction could be demonstrated.     

Inhibition of dopamine-β-hydroxylase by cytokinins

The activity of cell-free preparations of dopamine--hydroxylase from a mammalian source was inhibited by a number of N⁶-substituted adenine derivatives that are hormonally active as cytokinins in plant systems. The synthetic cytokinin N⁶-cyclohexylmethyladenine exhibited inhibitory activity equivalent to that of 1-phenyl-3-(2-thiazolyl)-2-thiourea (PTTU), a compound known to be a potent inhibitor of dopamine--hydroxylase activity. PTTU itself was found to exhibit cytokinin activity in the tobacco callus bioassay and to inhibit the activity of the plant enzyme, cytokinin oxidase. The possible significance of these observations is discussed in relation to known effects of cytokinins on phenethylamine metabolism.     

Plasma dopamine β-hydroxylase species dependence and the in vitro influence of NEM,copper, and pH

We have applied a sensitive radiochemical assay to measure DBH activities in several species at their pH optima and to estimate the effects of copper and N-ethylmaleimide (NEM) in the assay. The enzyme incubation was performed at 37° with 5 μl of diluted plasma and 5 × 10⁻⁴m [¹⁴C]tyramine in 0.17 m sodium acetate at pH 4.0–6.0. Activities were determined from the formation of [¹⁴C]octopamine, after periodate oxidation and extraction, measured by using liquid scintillation counting. Regardless of species, maximal activities could be obtained at low substrate concentrations only by adding 3.5 to 10 μm copper to the assay. Following the addition of NEM, DBH activities were suboptimal. Incremental NEM additions to a spectrophotometric assay at saturating tyramine concentrations resulted in maximal activity only in human plasma. With optimal copper in the radioenzymatic assay, representative human values ranged from 2.1 ± 0.2 to 7.4 ± 0.4 nmole/min/ml of plasma (mean ± SEM) at pH 5.5. Rat, rabbit, and dog ranged 100-fold less in activity and varied with respect to pH optimum. Studies with purified internal standards defined the influence of copper and NEM on endogenous inhibitor neutralization. Our results support the proposal that precise determinations of plasma DBH activities are limited by conditions within the assay.     

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.     

Specificity in steroid histochemistry,with special reference to the use of steroid solvents. distribution of 11 β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse

Summary Several factors influencing the steroiddehydrogenase histochemistry were investigated: diffusion of enzyme; inactivation of enzyme; effects of the steroid solvents commonly used; the validity in localization of the enzyme activity; nothing dehydrogenase reaction. 1. The importance in controlling the diffusion of each enzyme system to be studied is emphasized. Provided that the presence of SH-groups in the active centre of the dehydrogenase can be proved, a control experiment using a double-section incubation method should be carried out. 2. A comparison between the use of unfixed and briefly prefixed sections is recommended in order to avoid a possible distortion of the tissue during the incubation. The influence of prefixation on diffusion of enzymes or reaction products as well as on inactivation of enzymes must be studied. 3. The steroid solvents—especially dimethyl formamide caused a morphological distortion, and an inactivation and/or extraction of reaction products (the red monoformazan) in fresh frozen sections, these solvents should therefore be handled with caution. A special mixture of dimethyl formamide and propylene glycol is recommended. 4. The steroid should be completely soluble in the incubation medium in order to secure zero order kinetics. 5. Avoidance of sulphydryl nothing dehydrogenase reaction, since the reaction predominantly manifests itself as a red formazan obscuring sites with low dehydrogenase activity. 6. The localization of the NAD(P)H oxidase systems must be controlled, in order to ensure that they should not be a limiting factor in the detection of the dehydrogenase activity. Secondly, this investigation may act as a control on diffusion of dehydrogenase and/or reduced coenzyme. 7. That the investigation of the incubation time needed for initial visual reaction allows a certain quantitative estimation of the concentration of enzyme localized at different sites in the same section. The investigation should also include the red formazan, since it has recently been proved to be an intermediary step in the enzymic reduction of Nitro BT, and as such may reflect sites with low enzyme concentration.Further, some of the functional aspects of the activity of 11-hydroxysteroiddehydrogenaseNAD(P)H oxidase systems in the thymus were discussed, and lastly the localization of these systems in the kidney was revised.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.