Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017alpha.

The cDNA encoding cytochrome P-45017alpha from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

Cytochrome P450-dependent renal arachidonic acid metabolism in desoxycorticosterone acetate-salt hypertensive mice.

Cytochrome P450 (P450)-dependent arachidonic acid metabolites may act as mediators in the regulation of vascular tone and renal function. We studied arachidonic acid hydroxylase activities in renal microsomes from normotensive NMRI mice, desoxycorticosterone acetate (DOCA)-salt hypertensive mice, and DOCA-salt mice treated with either lovastatin or bezafibrate, both of which improve hemodynamics in this model. Control renal microsomes had arachidonic acid hydroxylase activities of 175+/-12 pmol. min(-1). mg(-1). The metabolites formed were 20- and 19-hydroxyarachidonic acid, representing approximately 80% and approximately 20% of the total hydroxylation. Treatment with DOCA-salt resulted in significantly decreased hydroxylase activities (to 84+/-4 pmol. min(-1). mg(-1)) of the total microsomal P450 content and a decrease in immunodetectable Cyp4a proteins. Lovastatin had no effect on these variables, whereas bezafibrate increased arachidonic acid hydroxylase activities to 163+/-12 pmol. min(-1). mg(-1). In situ hybridization with probes for Cyp4a-10, 12, and 14 revealed that Cyp4a-14 was the P450 isoform most strongly induced by bezafibrate. The expression was concentrated in the cortical medullary junction and was localized predominantly in the proximal tubules. In conclusion, these results suggest that the capacity to produce 20-hydroxyarachidonic acid is impaired in the kidneys of DOCA-salt hypertensive mice. Furthermore, bezafibrate may ameliorate hemodynamics in this model by restoring P450-dependent arachidonic acid hydroxylase activities. Lovastatin, on the other hand, exerts its effects via P450-independent mechanisms.

Inhibition of pressure natriuresis in mice lacking the AT2 receptor.

UNLABELLED: Inhibition of pressure natriuresis in mice lacking the AT2 receptor. BACKGROUND: Angiotensin II type 2 (AT2) receptor knockout mice have higher blood pressures than wild-type mice; however, the hypertension is imperfectly defined. We tested the hypothesis that renal mechanisms could be contributory. METHODS: We conducted pressure-natriuresis-diuresis experiments, measured renal cortical and medullary blood flow by laser Doppler methods, and explored cytochrome P450-dependent arachidonic acid metabolism by means of reverse transcription-polymerase chain reaction. RESULTS: Blood pressure was 15 mm Hg higher in AT2 receptor knockout mice than in controls, and pressure diuresis and natriuresis curves were shifted rightward. At similar renal perfusion pressures (113 to 118 mm Hg), wild-type mice excreted threefold more sodium and water than AT2 receptor knockout mice. Fractional sodium and water excretion curves were shifted rightward in parallel. Renal blood flow ranged between 6.72 and 7.88 mL/min/g kidney wet weight (kwt) in wild-type and between 5.84 and 6.15 mL/min/g kwt in AT2 receptor knockout mice. Renal vascular resistance was increased in AT2A receptor knockout mice. Cortical blood flow readings leveled at 2.5 V in wild-type and 1.5 V in AT2 receptor knockout mice. Medullary blood flow readings ranged between 0.8 and 1.0 V and increased 116% in wild-type mice as renal perfusion pressure was increased. This increase did not occur in AT2 receptor knockout mice. The glomerular filtration rate (GFR) was similar in both groups at approximately 1 mL/min/g kwt. Renal microsomes from AT2 receptor knockout mice had less activity in hydroxylating arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-meter) than controls, whereas renal AT1 receptor gene expression was increased in AT2 receptor knockout mice. CONCLUSIONS: Hemodynamic and tubular factors modify renal sodium handling in AT2 receptor knockout mice and may cause hypertension. AT2 receptor disruption induces alterations of other regulatory systems, including altered arachidonic acid metabolism, that may contribute to the intrarenal differences observed between AT2 receptor knockout and wild-type mice.

Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties.

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.

The alkane-hydroxylating enzyme system of the yeast Candida guilliermondii.

The growth of the investigated Candida guilliermondii strain on n-alkanes induces an alkane-hydroxylating enzyme system, which consists of a cytochrome P-450 and a NADPH-dependent reductase. The cytochrome P-450 was purified to 4 nmoles per mg protein. Long-chain alkanes, preferably hexadecane to octadecane, are hydroxylated to the corresponding primary alcohol by this enzyme system. The substrate induces a type I spectrum, other compounds checked type II spectra.

Cytochrome P-450 and alkane hydroxylase activity in Candida guilliermondii.

In the investigated Candida guilliermondii strain after growth on n-alkanes as the only carbon and energy source 5--10 nMol cytochrome P-450 per g cells (wet weight) could be detected. Cytochrome P-450 and alkane hydroxylase activity was found in the 100 000 xg pellet. Cofactor studies and inhibition experiments revealed the existence of a NADPH-dependent cytochrome P-450 alkane hydroxylase system.