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

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonastestosteroni 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 3α-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 Escherichiacoli. Rep1 does not bind to DNA but may bind to 3α-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3α-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.     

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.     

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5

Natural and synthetic steroid hormones excreted into the environment are potentially threatening the population dynamics of all kinds of animals and public health. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. 16S-rRNA analysis showed that bacterial strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the (3)H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5 h and 20 h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in adaptive steroid degradation in Comamonas testosteroni (C. testosteroni), in previous investigations, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established. By this meta-genomic system, two estradiol inducible genes coding 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively, which are involved in steroid degradation, were found in marine strain H5. In the present work, the 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli (E. coli) strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with isopropyl-β-D-thiogalactoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one step using nickel-chelate chromatography. After protein determination, 3-ketosteroid-delta-1-dehydrogenase (0.48 mg/ml) and carboxylesterase (1.28 mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In summary, we have shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA, and purified the corresponding proteins for further research. The exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. The strain might be used for the bioremediation of steroid contaminations in seawater.     

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.     

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.     

Understanding oligomerization in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: an in silico approach and evidence for an active protein

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni belongs to the short chain dehydrogenase/reductase (SDR) protein superfamily and catalyzes the oxidoreduction of a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also mediates the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to the bioremediation of natural and synthetic toxicants by C. testosteroni. Crystallization and structure analysis showed that 3alpha-HSD/CR is active as a dimer. Dimerization takes place via an interface axis which has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure. For example, 3alpha/20beta-HSD from Streptomyces hydrogenans exhibits two main subunit interfaces arranged about two non-crystallographic two-fold axes which are perpendicular to each other and referred to as P and Q. This mode of dimerization is, however, sterically impossible in 3alpha-HSD/CR because of a 28 amino acids insertion into the classical Rossmann-fold motif between strand betaE and helix alphaF. This insertion is masking helices alphaE and alphaF, thus preventing the formation of a four helix bundle and enables the dimerization via a P-axis interface. This type of dimerization in SDRs has never been observed in a crystal structure so far. The aim of this study was to investigate whether the lack of this predominantly alpha-helical subdomain keeps 3alpha-HSD/CR to be an active enzyme and whether, by an in silico approach, the formation of a homotetramer or even a novel oligomerization mode can be expected. Redesign of this interface was performed on the basis of site directed mutagenesis and according to other SDR structures by an approach combining "in silico" and "wet chemistry". Simulations of sterical and structural effects after different mutations, by applying a combination of homology modelling and molecular dynamic simulations, provided an effective tool for extensive mutagenesis studies and indicated the possibility of tetramer formation of truncated 3alpha-HSD/CR. In addition, despite lacking the extra loop domain, mutant 3alpha-HSD/CR was shown to be active towards a variety of standard substrates.     

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ–Δ isomerase pathway

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3β-hydroxy-steroid dehydrogenase/Δ⁵–Δ⁴ isomerase (3β-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3β-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3β-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13α-methyl analogue. In contrast to previous studies of fungi with 3β-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one were metabolized solely through the putative 3β-HSD/isomerase pathway, indicating that a 17β-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3β-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.     

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3α-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 °C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3α-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.     

Expression and Assembly Mechanism of the Capsid Proteins of a Satellite Virus (XSV) Associated with Macrobrachium rosenbergii Nodavirus

The extra small virus (XSV) is a satellite virus associated with Macrobrachium rosenbergii nodavirus (MrNV) and its genome consists of two overlapping ORFs, CP17 and CP16. Here we demonstrate that CP16 is expressed from the second AUG of the CP17 gene and is not a proteinase cleavage result of CP17. We further expressed CP17 and several truncated CP17s (in which the N- or C-terminus or both was deleted), respectively, in Escherichia coli. Except for the recombinant plasmid CP17ΔC10, all recombinant plasmids expressed soluble protein which assembled into virus-like particles (VLPs), suggesting that the C-terminus is important for VLP formation.     

Human 11β-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11β-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichiacoli, 11β-HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11β-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E.coli and the yeast Pichiapastoris. Furthermore, we investigated if the codon for methionine 31 in human 11β-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E.coli nor with that expressed in P.pastoris. Hydrophobicity plots proved the hydrophobic nature of 11β-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.     

Truncated KCNQ1 mutant, A178fs/105, forms hetero-multimer channel with wild-type causing a dominant-negative suppression due to trafficking defect

We identified a novel mutation Ala178fs/105 missing S3-S6 and C-terminus portions of KCNQ1 channel. Ala178fs/105-KCNQ1 expressed in COS-7 cells demonstrated no current expression. Co-expression with wild-type (WT) revealed a dominant-negative effect, which suggests the formation of hetero-multimer by mutant and WT. Confocal laser microscopy displayed intracellular retention of Ala178fs/105-KCNQ1 protein. Co-expression of the mutant and WT also increased intracellular retention of channel protein compared to WT alone. Our findings suggest a novel mechanism for LQT1 that the truncated S1-S2 KCNQ1 mutant forms hetero-multimer and cause a dominant-negative effect due to trafficking defect.     

Expression and NNK reducing activities of carbonyl reductase and 11β-hydroxysteroid dehydrogenase type 1 in human lung

The tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), which is found in high amounts in tobacco products, is believed to play an important role in lung cancer induction in smokers. NNK requires metabolic activation by cytochrome P450 mediated α-hydroxylation to exhibit its carcinogenic properties. On the other hand, NNK is inactivated by carbonyl reduction to its alcohol-equivalent 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronidation and final excretion into urine or bile. Carbonyl reduction and α-hydroxylation are the predominant pathways in man, and it has been postulated that the extent of these competing pathways determines the individual susceptibility to lung cancer. Moreover, only a minor part of all habitual smokers develop lung cancer, suggesting the existence of susceptibility genes. Microsomal 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) (EC 1.1.1.146) and cytosolic carbonyl reductase (CR) (EC 1.1.1.184) have been shown to be mainly responsible for NNAL formation in liver and lung. In the present study, we performed comparative investigations of human lung tissue samples from several patients with respect to the expression and activity of 11β-HSD 1 and carbonyl reductase. We observed varying levels in 11β-HSD 1 and carbonyl reductase expression in these patients, as revealed by RT-PCR and ELISA. Also, the tissue samples showed a different activity and inhibitor profile for both enzymes. According to our results, variations in the expression and activity of NNK carbonyl reducing enzymes may constitute a major determinant in the overall NNK detoxification capacity and thus may be linked to the great differences observed in the individual susceptibility of tobacco-smoke related lung cancer.     

The C-Terminus of ClpC1 of Mycobacterium tuberculosis Is Crucial for Its Oligomerization and Function

Mycobacterium tuberculosis ClpC1 is a member of the Hsp100/Clp AAA+ family of ATPases. The primary sequence of ClpC1 contains two N-terminal domains and two nucleotide binding domains (NBD). The second NBD has a long C-terminal sub-domain containing several motifs important for substrate interaction. Generally, ClpC proteins are highly conserved, however presence of C-terminal domains of variable lengths is a remarkable difference in ClpC from different species. In this study, we constructed deletion mutants at the C-terminus of M. tuberculosis ClpC1 to determine its role in the structure and function of the protein. In addition, a deletion mutant having the two conserved N-terminal domains deleted was also constructed to investigate the role of these domains in M. tuberculosis ClpC1 function. The N-terminal domains were found to be dispensable for the formation of oligomeric structure, and ATPase and chaperone activities. However, deletions beyond a specific region in the C-terminus of the ClpC1 resulted in oligomerization defects and loss of chaperonic activity of the protein without affecting its ATPase activity. The truncated mutants, defective in oligomerization were also found to have lost the chaperonic activity, showing the formation of oligomer to be required for the chaperonic activity of M. tuberculosis ClpC1. The current study has identified a region in the C-terminus of M. tuberculosis ClpC1 which is essential for its oligomerization and in turn its function.     

DNA-Unwinding Dynamics of Escherichia coli UvrD Lacking the C-Terminal 40 Amino Acids

The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3′ single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.