Terminal Hydroxylation Research Articles

The cytochrome P450 IV family: constitutive and inducible haemoproteins

The cytochrome P450 IV family: constitutive and inducible haemoproteins

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450alk (P450alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450alk. The coordinate expression of the P450alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450alk monooxygenase activity about 2-fold.

Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (Al) of a new P450 gene family (LII).

Specific induction of lauric acid ω‐hydroxylase by clofibrate, diethylhexyl‐phthalate and 2,4‐dichlorophenoxyacetic acid in higher plants

AbstractRecently, we have found in plant microsomes two laurate hydroxylases that catalyze the terminal hydroxylation or the in‐chain hydroxylation of the fatty acid. These two hydroxylases, which are both cytochrome P‐450 enzymes, are never found in the same plant. This study shows that the hypolipidemic drug clofibrate induces the lauric acid ω‐hydroxylase activity inVicia and soybean seedlings. The marked increase in activity (>20‐fold) produced by clofibrate was dose‐dependent but was not paralleled by an enhancement of bulk cytochrome P‐450 or cinnamic acid 4‐hydroxylase. Compounds related to clofibrate by structure (2,4‐dichlorophenoxyacetic acid) or effects (diethylhexyl‐phthalate) also stimulated the ω‐hydroxylating system in these plants. In contrast, the lauric acid in‐chain hydroxylase from Jerusalem artichoke tubers was induced less than cinnamic acid 4‐hydroxylase activity and bulk cytochrome P‐450 in tissues incubated in clofibrate solution. This suggests that clofibrate induces preferentially the laurate ω‐hydroxylating isozyme in plants.

The distinction of different types of cytochromes P-450 from the yeasts Candida tropicalis and Saccharomyces uvarum

The distinction between two types of cytochromes P-450 originating from microsomes of Candida tropicalis grown on glucose and on alkane was achieved. Criteria of differentiation between these two cytochrome P-450 forms were based on the characteristics of reduced carbon monoxide difference spectra, on substrate specificity, and on binding and inhibition kinetics of the fungistatic compound propiconazole. One cytochrome P-450 form catalyzed the 14α-demethylation of lanosterol and bound propiconazole with an equimolar ratio. This form was present in microsomes from glucose-grown cells and shared similar characteristics with the cytochrome P-450 originating from Saccharomyces uvarum grown on the same carbon source. The other cytochrome P-450 form catalyzed the terminal hydroxylation of aliphatic hydrocarbons and showed a less specific binding ratio with propiconazole (10 3 mol propiconazole for 1 mol cytochrome P-450). This type of cytochrome P-450 was only present in the microsomes of C. tropicalis grown on alkane.

Chemical and stereochemical aspects of propranolol metabolism. Diastereomeric 1-(1-hydroxy-2-propylamino)-3-(1-naphthoxy)-2-propanols produced by rat liver microsomal omega-hydroxylation.

A new metabolic pathway of terminal hydroxylation (omega-hydroxylation) of the N-isopropyl group of propranolol (1) was established. Selected ion-monitoring GC-MS analysis, based on use of the synthesized mixture of diastereoisomers of 1-(1-hydroxy-2-propylamino)-3-(1-naphthoxy)-2-propanol (2) as a standard, established formation of both diastereoisomers of 2 as metabolites of 1. These diastereoisomers were formed in unequal amounts when 1, its hexadeuterated analogue 8 or heptadeuterated analogue 9, were incubated in the presence of the rat liver microsomal fraction. Authentic (2R,2"S)-2, obtained from the amide formed from (2S)-3-(1-naphthoxy)-2-hydroxypropionic acid [(2S)-5] and (2S)-alaninol by diborane reduction, facilitated examination of stereochemical aspects of this process. From incubations of the enantiomers of 1 and pseudoracemic propranolol [equimolar (2R)-propranolol-3,3-d2 and (2S)-propranolol-d0] in the presence of the rat liver microsomal fraction, we established that the diastereomeric products were formed in the order (2S,2"S)-2 approximately equal to (2S,2"R)-2 greater than (2R,2"R)-2 greater than (2R,2"S)-2. (2S)-1, which was metabolized to 2 to a greater extent than (2R)-1, showed no stereoselectivity, affording about equal amounts of (2S,2"S)-2 and (2S,2"R)-2. (2R)-1, which was metabolized to 2 to a lesser extent, afforded considerably more (2R,2"R)-2 than (2R,2"S)-2. omega-Hydroxylation was a minor metabolic pathway in the microsomal incubation. About 2000X less 2 than 1-amino-3-(1-naphthoxy)-2-propanol (3), the product of N-dealkylation of 1, was formed.

Immunological probe of estrogen biosynthesis. Evidence for the 2 beta-hydroxylative pathway in aromatization of androgens.

The terminal hydroxylation in placental estrogen biosynthesis from androgens is at the 2 beta position. The 2 beta-hydroxy-19-oxoandrogen derivative collapses nonenzymatically to estrogen and is therefore the proximate precursor of the female hormone. To establish the role of this pathway in biological aromatization, an immunological approach was employed in which an antibody was obtained which recognizes 2 beta-hydroxy-19-oxygenated androgens but not intermediates oxygenated at C-19 only. Binding of the 2 beta-hydroxy-19-oxo intermediate by the antibody stabilizes it so that its nonenzymatic transformation to estrogen is delayed and results in slower estrogen formation. When placental microsomes were incubated with [1,2-3H]androstenedione in the presence of the antibody antiserum, a 50% decrease in [3H]estradiol formation and 3H2O release was observed when compared with identical incubations containing normal rabbit serum alone. This inhibition is blocked when the antibody is inactivated by presaturation with 2 beta, 19-dihydroxyandrostenedione. Precipitation of immunoglobulins from the incubations followed by heating liberated the 2 beta-hydroxy-19-oxo intermediate (30%) from the antibody, and resulted in its nonenzymatic collapse to estrogen with concomitant release of 3H2O. Control normal rabbit serum or blocked antibody incubations did not show a similar increase in [3H]estradiol or 3H2O yields in the precipitate. Heat treatment (90 degrees C) of the antibody but not normal rabbit serum incubations resulted in a similar increase in [3H]estradiol and 3H2O yields. These results are consistent with the hypothesis that the final and rate-determining hydroxylation in aromatization of androgens is at the 2 beta position and that this pathway is the dominant, if not the sole, route of estrogen biosynthesis by placental aromatase. The antibody probe also permits the characterization of aromatization mechanisms in tissues other than the placenta.

Metabolism of C 19-radiosteroids by explants of canine prostate and epididymis with disposition as hydroxylated products: A possible mechanism for androgen inactivation

Explants from canine prostate and epididymis were used to study the metabolic disposition of labelled testosterone, 17β-hydroxy-5α-androstan-3-one (5α-DHT), 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol (3β-diol) in serum-free Trowell's T8 medium under organ-culture conditions which maintained the structural integrity of the tissues. The 1.7 μM and 8.5 nM radiotestosterone-derived uptake and chromatographically-determined disposition patterns were obtained after 2, 6 and 21 h in culture at 37°C. Prostatic uptake, metabolism and high-affinity binding to total 0.4 M KCl-extractable protein were compared after 21 h exposure to each of the four [ 3H]-substrates. A characteristic feature of radiotestosterone disposition was the accumulation of C 19O 3-metabolites in explants, and release of large quantities into the medium between 6 h and 21 h. Major components of the C 19O 3-steroids in 21 h media were partially identified as 5α-reduced 6-and (predominantly) 7-hydroxylated radiometabolites by CrO 3-oxidation and crystallization of 5α-androstane-3,6,17-trione and 5α-androstane-3,7,17-trione to constant SA. 850 nM estradiol inhibited prostatic formation of hydroxylation products from 8.5 nM [ 3H]-testosterone by 46%, with accumulation of 3β-hydroxy-5α-androstan-17-one and 3β-diol. The optimal substrate for the prostatic hydroxylation reactions was 3β-diol. It generated much less 5α-DHT in free and specifically-bound forms than the other substrates. The results suggest that the 3β-hydroxysteroid oxidoreductase-hydroxylase system provides a mechanism by which male accessory reproductive glands dispose of free and bound intracellular androgens. Estrogen inhibition of the terminal hydroxylation reactions points to a new mode of regulation of male hormone action.