Extracellular 3beta-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D

Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5-->delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Delta(1)-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Delta(1)-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Delta(1)-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

3-甾酮-1-脱氢酶基因的表达及甾体转化分析

本文利用带有P43启动子的表达分泌载体pWB980,实现了简单节杆菌3-甾酮-1-脱氢酶在枯草芽孢杆菌中的表达,表达出的目的蛋白的分子量为55KDa。利用分光光度法检测得到胞内和胞外可溶性部分的酶活分别为110±0.5mU和15±0.6mU每毫克蛋白, 比出发菌株简单节杆菌提高了将近30倍。重组芽孢杆菌对甾体底物4-AD的转化率为45.3％,比出发菌株简单节杆菌提高了近10倍。利用枯草芽孢杆菌对甾体底物进行脱氢为甾体药物的生产开辟了一个新的途径。     

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.     

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.     

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

Cholest-4-En-3-One-Δ1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ¹-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ¹-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ¹-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.     

Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum.

Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.     

Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5α-reductase

The location and some characteristics of rat adrenal C(19)-steroid 5alpha-reductase were investigated by using [7alpha-(3)H]androst-4-ene-3,17-dione and [7alpha-(3)H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3alpha- and 3beta-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17beta-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C(19)-steroid 5alpha-reductase, which showed similar values for K(m) and V(max.) when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5alpha-androstane-3,17-dione and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5alpha-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5alpha-reduction of C(19)- and C(21)-steroids in the rat adrenal.     

A New Bacterial Steroid Degradation Gene Cluster in Comamonas testosteroni TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Δ¹-dehydrogenase and 3-ketosteroid-Δ⁴(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

The biotransformation of hyodeoxycholic acid by Pseudomonas sp. NCIB 10590 under anaerobic conditions

The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6 alpha-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6 alpha-hydroxyandrosta-1,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-oxochola-1,4-diene-24-oic acid and 6 alpha-hydroxy-3-oxochola-1,4-diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,17-trione, androsta-1,4-diene-3,6,17-trione, androsta-1,4-diene-3,17-dione, 17 beta-hydroxyandrosta-1,4-diene-3-one and 6 alpha-hydroxyandrost-4-ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6 alpha-dehydroxylase enzyme. A biochemical pathway of hyodeoxycholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzyme.     

Characterization and application of fusidane antibiotic biosynethsis enzyme 3-ketosteroid-?1-dehydrogenase in steroid transformation

Microbial ?(1)-dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-?(1)-dehydrogenase (kstD(F)) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD(F) encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstD(F) in Escherichia coli and Pichia pastoris showed that all kstD(F) activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstD(F) was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71(I)) and GS115 (GS115(I)) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstD(F) was expressed intracellularly. Under the same conditions, KM71(I) was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115(I) could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17β-hydroxysteroid dehydrogenase in P. pastoris GS115(I), which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115(I) and KM71(I) may provide alternative means of preparing ADD or BD. In brief, we show here that kstD(F) is a promising enzyme in steroid ?(1)-dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.