A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.     

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.     

Allantoin racemase: a new enzyme from Pseudomonas species.

1. Allantoin racemase is a novel enzyme which catalyzes the conversion of S(+)-and R(minus)-allantoin into the racemate. 2. The enzyme is present in Pseudomonas testosteroni, Pseudomonas putida and five biotypes of Pseudomonas fluorescens, but absent in a number of other Pseudomonas species. 3. The enzyme of Ps. testosteroni was purified 133-fold and exposes optimal activity at pH 8.0-8.2 and 50 degrees C. The enzyme is stable on heating for 15 min at 70 degrees C. 4. The enzyme appeared to be specific for the optical isomers of allantoin and no cofactors are involved in the reaction. 5. The optical aspecificity of allantoinase of Proteus rettgeri was reaffirmed.     

Phthalate metabolism in Pseudomonas testosteroni: accumulation of 4,5-dihydroxyphthalate by a mutant strain.

A mutant strain of Pseudomonas testosteroni blocked in phthalate catabolism converted phthalate into 4,5-dihydroxyphthalate. The latter compound was isolated, and its physical properties were determined. A stoichiometric conversion of the compound to protocatechuate was demonstrated spectrophotometrically with crude extracts of a protocatechuate 4,5-dioxygenase-deficient mutant. Therefore, phthalate is metabolized through 4,5-dihydroxyphthalate and protocatechuate, which is further degraded by protocatechuate 4,5-dioxygenase in P. testosteroni. By using several mutants blocked in phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase was shown to be induced by phthalate. A simple spectrophotometric assay for the enzyme is also reported.     

Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling

Abstract Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans , and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus , this enzyme is quite different from that of R. acidophila . Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c ; Rhodopseudomonas acidophila     

The purification and properties of urocanase from Pseudomonas testosteroni.

Urocanase (urocanate hydratase, EC 4.2.1.49) purified from Pseudomonas testosteroni has a mol.wt. of 118000 determined by sedimentation-equilibrium analysis. Ultracentrifugation in 6M-guanidine hydrochloride and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate show that the enzyme consists of two identical or very similar subunits. It is, like urocanase isolated from other sources, inhibited by reagents that react with carbonyl groups. Although urocanase from Ps. testosteroni is strongly inhibited by NaBH4, no evidence could be obtained for the presence of covalently bound 2-oxobutyrate as a prosthetic group; this is in contrast with findings elsewhere for urocanase from Pseudomonas putida. Urocanase from Ps. testosteroni does not contain pyridoxal 5'-phosphate as a coenzyme and in this respect is similar to all urocanases studied in purified form.     

Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

Cloning and identification of a new repressor of 3,17β-Hydroxysteroid dehydrogenase of Comamonas testosteroni

Molecular Biology Reports - 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni....     

3α-羟类固醇脱氢酶基因的质粒载体构建和表达

目的 实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达.方法 从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达.提取细菌总蛋白进行SDS-PAGE分析并测定酶活性.结果 经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍.结论 该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础.     

Gentisate 1,2-dioxygenase from pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans

The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P. acidovorans and P. testosteroni, the two divergent species of the acidovorans group of Pseudomonas. Both enzymes exhibit a 40-fold higher specific activity than previous preparations and have an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 and 158,000, respectively). The enzymes have different amino terminal sequences, amino acid contents, and isoelectric points. Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively to give an EPR active complex (S = 3/2), showing that the iron is Fe2+ with coordination sites for exogenous ligands. The EPR spectra of these complexes are altered uniquely for each enzyme when gentisate is bound. This suggests that substrate binds to or near the iron and shows that the substrate-iron interactions of each enzyme are subtly different. The kinetic parameters for turnover of gentisate by the enzymes are nearly identical (kcat/Km = 4.3 x 10(6) s-1 M-1). Both enzymes cleave a wide range of gentisate analogs substituted in the 3 or 4 ring position, although at reduced rates relative to gentisate. Of the two enzymes, P. testosteroni gentisate 1,2-dioxygenase exhibits substantially lower kcat/Km values for the turnover of these compounds. Evidence for both steric and electronic substituent effects is obtained. In accord with the results of Wheelis et al. (Wheelis, M. L., Palleroni, N. J., and Stanier, R. Y. (1967) Arch. Mikrobiol. 59, 302-314), 3-hydroxybenzoate is shown to be metabolized by P. acidovorans through the gentisate pathway, and gentisate 1,2-dioxygenase is the only ring cleavage dioxygenase induced. In contrast, 3-hydroxybenzoate is metabolized by P. testosteroni exclusively through the protocatechuate pathway utilizing protocatechuate 4,5-dioxygenase, although gentisate 1,2-dioxygenase is coinduced. Growth of P. testosteroni on 3-O-methylbenzoate or 5-O-methylsalicylate is shown to result in a approximately 10-fold increase in the amount of gentisate 1,2-dioxygenase relative to protocatechuate 4,5-dioxygenase. Together, these results suggest that induction of gentisate 1,2-dioxygenase by 3-hydroxybenzoate in P. testosteroni may be adventitious and that this enzyme may function in fundamentally different metabolic pathways in the two related Pseudomonas species.     

Regulation of testosterone degradation in Comamonas testosteroni

Recently, we have identified a gene encoding a LuxR-type factor, TeiR (Testosterone-inducible Regulator), which positively regulates steroid degradation in Comamonas testosteroni. Herein, we demonstrate that TeiR interacts in vivo with steroid catabolic gene promoters. The presence of testosterone induces a significant TeiR protein increase at the early logarithmic phase of growth. Interestingly, it is not until the early stationary phase where the activation of a steroid-inducible gene promoter is observed, indicating that testosterone might not be the true inductor of the steroid degradation pathway. In addition, beta-galactosidase expression driven by a testosterone-inducible promoter is prematurely activated in cells cultured in medium supplemented with ethyl acetate extracts obtained from the early stationary phase cell-free supernatants of C. testosteroni grown in presence of testosterone. Complementation experiments of C. testosteroni wild type performed with teiR deletion constructs indicate that extra-copies of deleted-TeiR exert a dominant negative effect on the wild-type TeiR protein. While, when C. testosteroni teiR mutants were used to carry out complementation assays only the full length gene can overcome the teiR mutant phenotype. Altogether these findings indicate that TeiR regulates steroid catabolic genes interacting with their promoters and suggest that this interaction requires the presence of a testosterone-derived metabolite to induce the system.     

2-pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in Pseudomonas species.

2-Pyrone-4,6-dicarboxylate hydrolase was purified from 4-hydroxybenzoate-grown Pseudomonas testosteroni. Gel filtration and electrophoretic measurements indicated that the preparation was homogeneous and gave a molecular weight of 37,200 for the single subunit of the enzyme. Hydrolytic activity was dependent upon a functioning sulfhydryl group(s) and was freely reversible; the equilibrium position was dependent upon pH, with equimolar amounts of pyrone and open-chain form present at pH 7.9. Since the hydrolase was strongly induced when the nonfluorescent organisms P. testosteroni and P. acidovorans grew with 4-hydroxybenzoate, it is suggested that 2-pyrone-4,6-dicarboxylate is a normal intermediate in the meta fission degradative pathway of protocatechuate. Laboratory strains of fluorescent pseudomonads did not metabolize 2-pyrone-4,6-dicarboxylate, but a strain of P. putida was isolated from soil that utilized this compound for growth; the hydrolase was then induced, but it was absent from extracts of 4-hydroxybenzoate-grown cells that readily catabolized protocatechuate by ortho fission reactions. 2-Pyrone-4,6-dicarboxylic acid was the major product formed when gallic acid was oxidized by purified protocatechuate 3,4-dioxygenase. Protocatechuate 4,5-dioxygenase gave only the open-chain ring fission product when gallic acid was oxidized, but the enzyme attacked 3-O-methylgallic acid, giving 2-pyrone-4,6-dicarboxylic acid as the major product. Cell suspensions of 4-hydroxybenzoate-grown P. testosteroni readily oxidized 3-O-methylgallate with accumulation of methanol.     

Inhibition of microbial cholesterol oxidases by dimethylmorpholines

Cholesterol oxidase is a potentially important enzyme in steroid transformations, catalysing the conversion of 3-hydroxy-5-ene steroids to 3-keto-4-ene derivatives via a 3-keto-5-ene intermediate. Morpholine derivatives, especially fenpropimorph and tridemorph, were found to block selectively the isomerisation activity of cholesterol oxidases isolated from Nocardia erythropolis, Streptomyces sp., Pseudomonas testosteroni and Schizophyllum commune. These enzymes differ strongly in physical characteristics and catalytic behaviour. The effectiveness of the inhibitors varied with the cholesterol oxidase tested. Fenpropimorph was most effective with each of the 4 enzymes, 50 mg/l inhibiting about 50% of the enzyme activity. Inhibition was instantaneous and followed a reversible competitive mechanism in Streptomyces sp. and a reversible non-competitive mechanism in Nocardia erythropolis and Schizophyllum commune. An irreversible type of inhibition was observed for P. testosteroni cholesterol oxidase.     

Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni.

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.