Molecular biology of rat steroid 11 beta-hydroxylase [P450(11 beta)] and aldosterone synthase [P450(11 beta, aldo)].

The molecular features of rat steroid 11ß-hydroxylase [P450(11ß)] and aldosterone synthase [P450(11ß, aldo)] are discussed. P450(11ß) is biosynthesized as a precursor form composed of 499 amino acids, having a 24-amino acid extension peptide. Two species of P450(11ß, aldo) were identified; a precursor form of P450(11ß, aldo)-1 is 510 amino acids long and has a 34-amino acid extension peptide, while that of P450(11ß, aldo)-2 is 500 amino acids long and has a 24-amino acid extension peptide. The 286th amino acid of P450(11ß, aldo)-1 is Glu, while that of P450(11ß, aldo)-2 is Lys. The cDNA-expression studies showed that P450(11ß, aldo)-1 had the aldosterone producing activity whereas P450(11ß, aldo)-2 had no activity, suggesting that Glu286 of P450(11ß, aldo) plays an important role in the catalysis. The amino acid sequence of a region in P450(11ß) from Leu337 through Pro352 is highly conserved among the steroidogenic P450s. Functional expression studies on the cDNAs for two P450(11ß)s showed that P450(11ß) catalyzes the 11ß-, 18- and 19-hydroxylations of 11-deoxycorticosterone, but not the aldosterone synthesis. P450(11ß, aldo), on the other hand, catalyzes the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. The two P450(11ß)s were also shown to catalyze the conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol and cortisone.     

Functional expression of the cDNAs encoding rat 11 beta-hydroxylase [cytochrome P450(11 beta)] and aldosterone synthase [cytochrome P450(11 beta, aldo)]

Expression plasmids containing two cDNAs of a rat cytochrome P450(11 beta) family, pcP450(11 beta)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21-26] and pcP450(11 beta, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245-252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11 beta) and P450(11 beta, aldo) were determined by using 11-deoxycorticosterone, corticosterone, 18-hydroxy-11-deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-deoxycorticosterone as a substrate. Cytochrome P450(11 beta) catalyzed 11 beta-, 18- and 19-hydroxylations of 11-deoxycorticosterone and 19-oxidation or 19-hydroxy-11-deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11 beta, aldo) catalyzed 11 beta- and 18-hydroxylations of 11-deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-deoxycorticosterone was catalyzed by cytochrome P450(11 beta, aldo).     

Molecular cloning and expression of cDNAS encoding rat aldosterone synthase: variants of cytochrome P-450(11 beta)

Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-450(11 beta),aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-450(11 beta),aldo-2) had an extension peptide identical to that of rat P-450(11 beta). Glu at the 320th position of P-450(11 beta),aldo-1 was replaced with Lys in P-450(11 beta),aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-450(11 beta). Constructed expression vector containing the cDNA for extension peptide of P-450(11 beta) and the mature protein of P-450(11 beta),aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

Functional expression of cDNAs for bovine 11 beta-hydroxylase-aldosterone synthases, P450(11 beta)-2 and -3 and their chimeras.

Two molecular species of bovine P450(11β), P450(11β)-2 and P450(11β)-3 have been identified, in which the amino acid differences were found at the 6th, 36th and 82nd positions from the NH₂-termini of the mature proteins. They catalyzed the 11β-, 18- and 19-hydroxylation and aldosterone formation from 11-deoxycorticosterone, and the rate of production of 18-hydroxycorticosterone and aldosterone by P450(11β)-3 was greater than that by P450(11β)-2 [Morohashi et al., J. Biochem. 107 (1990) 635–640].

In this study, chimeric clones were constructed whose 6th, 36th and 82nd amino acid residues were exchanged with each other. Two original clones and six chimeric clones were expressed in COS-7 cells, and their steroidogenic activities studied. The ratio of aldosterone or 18-hydroxycorticosterone production to corticosterone production by one clone was compared with that of the other. The ratios for the four clones having Gly36 [P450(11β)-3 type] were 0.08–0.22, whereas those for the clones having Ser36 [P450(11β)-2 type] were 0.03–0.05, suggesting that the Gly36 structure is important for aldosterone production.     

Cytochrome P450(11β): Structure-function relationship of the enzyme and its involvement in blood pressure regulation

Cytochrome P450(11β) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11β) family enzymes suggested that the enzymes having both 11β-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11β-hydroxylases of these animals. (2) The molecular nature of the P450(11β) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11β) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11β-, 18- and 19-Hydroxylation activities of adrenal mitochondria prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.     

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with β-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with β-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56 000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (M_r 55 000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identify with cod cytochrome P-450b (M_r 54 000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

The synthesis of P-(2-acetamido-2-deoxy-α-D-glucopyranosyl) P-ficaprenyl pyrophosphate

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α,β-D-glucopyranosylammonium phosphate was prepared by the action of crystalline phosphoric acid on 2-acetamido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The α-D anomer (3) was the main product, and was isolated pure by preparative thin-layer chromatography or by removal of the β-D anomer (6) by selective acid hydrolysis. Ficaprenyl phosphate was prepared from ficaprenol, obtained as an isomeric mixture (mainly C₅₅) from an extract of Ficus elastica. Compound 3 was converted into the free acid and then into the tributyl-ammonium salt, which was treated with P¹-diphenyl P²-ficaprenyl pyrophosphate to give the acetylated pyrophosphate diester 8, characterized by analytical, spectral, and hydrogenolytic studies. Deacetylation of 8 gave the synthetic “lipid intermediate”, P¹-(2-acetamido-2-deoxy-D-glucopyranosyl) P²-ficaprenyl pyrophosphate (9), the properties of which were compared with those of natural substances considered to be active in the biosynthesis of teichoic acids.     

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodlogy of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulos) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450₉. It has been proven to be different from all preceedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450₉, does not recognize rat liver microsomes; thus this cytochrome P-450₉ is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quatitative polymorphism. In reconstituted system, cytochrome P-450₉ is able to hydroxylate all substrates tested but is not specific on any; its exatc role in xenobiotic metabolism in man remains to be elucidated.     

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.     

地黄C3H基因的克隆及生物信息学分析

香豆酸-3-羟化酶属于植物中最大的蛋白酶细胞色素P450家族之一,在植物生命活动中发挥着重要作用。为了解地黄香豆酸-3-羟化酶基因RgC3H合成毛蕊花糖苷的功能,该研究基于地黄代谢组学分析获得KEGG途径中的C3H,采用多重比对在NCBI中获得同源基因的一个保守序列,并基于该保守序列和地黄SRA数据库,采用电子克隆和RT-PCR克隆技术获得地黄C3H基因全长CDS(RgC3H),对其进行生物信息学分析。结果表明:RgC3H基因全长为1 530 bp,且编码一个含509个氨基酸、分子量为57.91 kD、无信号肽的蛋白质; 基于氨基酸序列的结构分析显示,RgC3H有一个保守区域-P450结构域; 系统进化分析结果显示,RgC3H与芝麻和猴面花的C3H基因具有很高的同源性。上述结果为进一步研究RgC3H基因在地黄毛蕊花糖苷生物合成途径中的作用奠定了基础。     

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH₂-terminal sequence and catalytic activity of them were determined. The NH₂-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH₂-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetmaine, 450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 ad P450 UT-2 were active in the 2α- and 16α-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6β-, 7α- and 15α-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.     

Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-H8]-analogues through a common synthetic route

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-³H₈]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10

The nitrilases from Fusarium solani O1 and Aspergillus niger K10 showed a broad substrate specificity for carbocyclic and nonaromatic heterocyclic amino nitriles, the preferred substrates being five-membered γ-amino nitrile (±)-1a, six-membered γ-amino nitriles (±)-3a, (±)-5a and (±)-6a, pyrrolidine-3-carbonitriles (±)-9a and (±)-10a as well as piperidine-4-carbonitriles 14a and 15a. Both enzymes showed a strong diastereopreference for cis- vs. trans-γ-amino nitriles. The electronic and steric effects of N-protecting groups affected the reactivity of the nitriles. Amides as by-products of the nitrilase-catalyzed reaction were produced from heterocyclic amino nitriles (±)-9a, (±)-10a, 14a and 15a by the A. niger enzyme but only from nitrile (±)-9a by the F. solani enzyme.     

An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Production of 19-oic-11-deoxycorticosterone from 19-oxo-11-deoxycorticosterone by cytochrome P-450(11)beta and nonenzymatic production of 19-nor-11-deoxycorticosterone from 19-oic-11-deoxycorticosterone

19-Oxo-11-deoxycorticosterone was incubated with a cytochrome P-450(11)beta-reconstituted system, and the metabolites were analyzed by high performance liquid chromatography(HPLC). The main product found after chromatography was collected and treated with diazomethane. HPLC and 1H-NMR analysis of the methylated derivative indicated that it was 19-oic-11-deoxycorticosterone methyl ester. When 19-oic-11-deoxycorticosterone was stored at -20 degrees C for 1 month, it was spontaneously converted to other steroids. Structural analysis of the main degradation product indicated that it was 19-nor-11-deoxycorticosterone. These results suggest that the conversion of 19-oxo-11-deoxycorticosterone to 19-oic-11-deoxycorticosterone occurs through the P-450(11)beta-catalyzed reaction, and that the 19-oic-11-deoxycorticosterone thus formed is nonenzymatically converted into 19-nor-11-deoxycorticosterone.