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Edmund Maser - One of the best experts on this subject based on the ideXlab platform.
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Genome Sequence of Comamonas testosteroni ATCC 11996, a Representative Strain Involved in Steroid Degradation
Journal of bacteriology, 2012Co-Authors: Wenjie Gong, Michael Kisiela, Markus B. Schilhabel, Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.
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testosterone inducible regulator is a kinase that drives steroid sensing and metabolism in Comamonas testosteroni
Journal of Biological Chemistry, 2008Co-Authors: Andre Gohler, Guangming Xiong, Simone Paulsen, Gabriele Trentmann, Edmund MaserAbstract:The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.
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characterization and recombinant expression of the translational repressor repb of 3α hydroxysteroid dehydrogenase carbonyl reductase in Comamonas testosteroni
Chemico-Biological Interactions, 2003Co-Authors: Guangming Xiong, Edmund Maser, Hans Jorg MartinAbstract:Abstract 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The gene of 3α-HSD/CR ( hsdA ) was cloned and characterized by our group. We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression. To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state. Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level. The addition of testosterone to the culture medium led to a derepression. Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA , but has no effect on hsdA , with which it happens to partly overlap. The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA. In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3α-HSD/CR.
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the crystal structure of 3alpha hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
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the crystal structure of 3α hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
Guangming Xiong - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of a novel repressor luxr in Comamonas testosteroni
Chemico-Biological Interactions, 2017Co-Authors: Tianyuan Pan, Yang Zhang, Guangming XiongAbstract:Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram negative bacterium which can use steroid as a carbon and energy source. 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme for the degradation of steroid hormones in C. testosteroni. The LuxR regulation family is a group of regulatory proteins which play important role in gram negative bacterium. The luxr gene is located on 58 kb upstream of 3,17β-HSD gene with the opposite transcription orientation in the chromosomal DNA of C. testosteroni. An open reading frame of this putative luxr gene consists of 1125 bp and is translated into a protein containing 374 amino acids. The luxr gene was cloned into plasmid pK18 and plasmid pK-LuxR1 was obtained. E. coli HB101 was co-transformed by pK-LuxR1 and pUC912-10, pUC1128-5 or pUC3.2-4 (which contain βhsd gene and different length promoter, repeat sequences). The result of ELISA showed that LuxR protein is a negative regulator for 3,17β-HSD expression. The luxr gene in C. testosteroni was knock-out by homologous integration. 3,17β-HSD expression was increased in the mutant (C.T.-L-KO1) comparing to that in wild-type C. testosteroni (C.T.) after 0.5 mM testosterone induction. The mutant C.T.-L-KO1 and wild-type C. testosteroni were cultured at 27 °C and 37 °C. The result of growth curve proved that LuxR has also effect on the bacterial growth.
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Genome Sequence of Comamonas testosteroni ATCC 11996, a Representative Strain Involved in Steroid Degradation
Journal of bacteriology, 2012Co-Authors: Wenjie Gong, Michael Kisiela, Markus B. Schilhabel, Guangming Xiong, Edmund MaserAbstract:Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.
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testosterone inducible regulator is a kinase that drives steroid sensing and metabolism in Comamonas testosteroni
Journal of Biological Chemistry, 2008Co-Authors: Andre Gohler, Guangming Xiong, Simone Paulsen, Gabriele Trentmann, Edmund MaserAbstract:The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.
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characterization and recombinant expression of the translational repressor repb of 3α hydroxysteroid dehydrogenase carbonyl reductase in Comamonas testosteroni
Chemico-Biological Interactions, 2003Co-Authors: Guangming Xiong, Edmund Maser, Hans Jorg MartinAbstract:Abstract 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The gene of 3α-HSD/CR ( hsdA ) was cloned and characterized by our group. We have also reported that two repressor proteins (RepA and RepB) have been identified which regulate hsdA expression. To further characterize RepB, the protein was expressed in Escherichia coli and purified in an active state. Gel shift experiments showed that RepB binds to a 16 nucleotide sequence downstream of AUG of the hsdA mRNA, providing evidence that RepB acts on the translational level. The addition of testosterone to the culture medium led to a derepression. Furthermore, a plasmid was prepared containing a point mutation that inactivates only repA , but has no effect on hsdA , with which it happens to partly overlap. The result of coexpression experiments with this construct and a plasmid containing the genetic information for RepB showed that RepB is still active and is therefore not dependent on a functional RepA. In conclusion, RepB is a novel regulatory protein that inhibits the translation of hsdA mRNA, thereby leading to a decreased expression of 3α-HSD/CR.
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functional expression purification and characterization of 3alpha hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni
Biochemical and Biophysical Research Communications, 2000Co-Authors: Edmund Maser, Eric Mobus, Guangming XiongAbstract: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.
Shuang-jiang Liu - One of the best experts on this subject based on the ideXlab platform.
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Chemotaxis Towards Aromatic Compounds: Insights from Comamonas testosteroni.
International journal of molecular sciences, 2019Co-Authors: Yun-hao Wang, Zhou Huang, Shuang-jiang LiuAbstract:Chemotaxis is an important physiological adaptation that allows many motile bacteria to orientate themselves for better niche adaptation. Chemotaxis is best understood in Escherichia coli. Other representative bacteria, such as Rhodobacter sphaeroides, Pseudomonas species, Helicobacter pylori, and Bacillus subtilis, also have been deeply studied and systemically summarized. These bacteria belong to α-, γ-, e-Proteobacteria, or Firmicutes. However, β-Proteobacteria, of which many members have been identified as holding chemotactic pathways, lack a summary of chemotaxis. Comamonas testosteroni, belonging to β-Proteobacteria, grows with and chemotactically responds to a range of aromatic compounds. This paper summarizes the latest research on chemotaxis towards aromatic compounds, mainly from investigations of C. testosteroni and other Comamonas species.
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developing a synthetic biology toolkit for Comamonas testosteroni an emerging cellular chassis for bioremediation
ACS Synthetic Biology, 2018Co-Authors: Qiang Tang, Shuang-jiang LiuAbstract:Synthetic biology is rapidly evolving into a new phase that emphasizes real-world applications such as environmental remediation. Recently, Comamonas testosteroni has become a promising chassis for bioremediation due to its natural pollutant-degrading capacity; however, its application is hindered by the lack of fundamental gene expression tools. Here, we present a synthetic biology toolkit that enables rapid creation of functional gene circuits in C. testosteroni. We first built a shuttle system that allows efficient circuit construction in E. coli and necessary phenotypic testing in C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further used a hybrid strategy to create engineered promoters to expand expression strength and dynamics. Additionally, we tested the T7 RNA Polymerase-PT7 promoter system and reduced its leaky expression through promoter mutation for gene expression. By coupling random library construction with FACS screening, we further developed a synthetic T7 pro...
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Engineering the bacterium Comamonas testosteroni CNB-1: Plasmid curing and genetic manipulation
Biochemical Engineering Journal, 2018Co-Authors: Qiang Tang, Shuang-jiang LiuAbstract:Abstract Comamonas species are attractive microbial hosts for bioremediation engineering due to their versatile metabolic capacity and diverse niches inhabited. To establish Comamonas testosteroni CNB-1—a common Comamonas strain—as a programmable cellular chassis, its indigenous 91-kb-long plasmid, pCNB1, must be eliminated; in addition, the organism has to exhibit feasiblity for effective genetic manipulation. Here we present a novel plasmid curing strategy involving rare-cutting homing endonuclease and a selection and counter-selection system. With a self-eliminating helper plasmid, we successfully removed the target plasmid pCNB1, along with the helper plasmid itself, in a single step at an efficiency of 64%. By enhancing the homing endonuclease expression, we were able to further improve the efficiency to almost 100%. The curing of pCNB1 provided a plasmid-free strain for hosting an IncP-type replicon based shuttle vehicle which can be utilized for gene circuit engineering. Additionally, using a PCR-based Cre-loxP system, we demonstrated an increased ability to genetically manipulate the chromosome of C. testosteroni CNB-1. This study provides a rapid and effective solution to eliminate indigenous plasmids, and also facilitates the development of C. testosteroni as a promising chassis for future remediation applications.
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Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation
2018Co-Authors: Qiang Tang, Shuang-jiang LiuAbstract:Synthetic biology is rapidly evolving into a new phase that emphasizes real-world applications such as environmental remediation. Recently, Comamonas testosteroni has become a promising chassis for bioremediation due to its natural pollutant-degrading capacity; however, its application is hindered by the lack of fundamental gene expression tools. Here, we present a synthetic biology toolkit that enables rapid creation of functional gene circuits in C. testosteroni. We first built a shuttle system that allows efficient circuit construction in E. coli and necessary phenotypic testing in C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further used a hybrid strategy to create engineered promoters to expand expression strength and dynamics. Additionally, we tested the T7 RNA Polymerase-PT7 promoter system and reduced its leaky expression through promoter mutation for gene expression. By coupling random library construction with FACS screening, we further developed a synthetic T7 promoter library to confer a wider range of expression strength and dynamic characteristics. This study provides a set of valuable tools to engineer gene circuits in C. testosteroni, facilitating the establishment of the organism as a useful microbial chassis for bioremediation purposes
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direct sensing and signal transduction during bacterial chemotaxis toward aromatic compounds in Comamonas testosteroni
Molecular Microbiology, 2016Co-Authors: Zhou Huang, Chengying Jiang, Rebecca E Parales, Shuang-jiang LiuAbstract:Micro-organisms sense and chemotactically respond to aromatic compounds. Although the existence of chemoreceptors that bind to aromatic attractants and subsequently trigger chemotaxis have long been speculated, such a chemoreceptor has not been demonstrated. In this report, we demonstrated that the chemoreceptor MCP2901 from Comamonas testosteroni CNB-1 binds to aromatic compounds and initiates downstream chemotactic signaling in addition to its ability to trigger chemotaxis via citrate binding. The function of gene MCP2901 was investigated by genetic deletion from CNB-1 and genetic complementation of the methyl-accepting chemotaxis protein (MCP)-null mutant CNB-1Δ20. Results showed that the expression of MCP2901 in the MCP-null mutant restored chemotaxis toward nine tested aromatic compounds and nine carboxylic acids. Isothermal titration calorimetry (ITC) analyses demonstrated that the ligand-binding domain of MCP2901 (MCP2901LBD) bound to citrate, and weakly to gentisate and 4-hydroxybenzoate. Additionally, ITC assays indicated that MCP2901LBD bound strongly to 2,6-dihydroxybenzoate and 2-hydroxybenzoate, which are isomers of gentisate and 4-hydroxybenzoate respectively that are not metabolized by CNB-1. Agarose-in-plug and capillary assays showed that these two molecules serve as chemoattractants for CNB-1. Through constructing membrane-like MCP2901-inserted Nanodiscs and phosphorelay activity assays, we demonstrated that 2,6-dihydroxybenzoate and 2-hydroxybenzoate altered kinase activity of CheA. This is the first evidence of an MCP binding to an aromatic molecule and triggering signal transduction for bacterial chemotaxis.
Ralf Ficner - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of 3alpha hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
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the crystal structure of 3α hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
Eric Mobus - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of 3alpha hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
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the crystal structure of 3α hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase reductase family
Journal of Biological Chemistry, 2000Co-Authors: Clemens Grimm, Edmund Maser, Eric Mobus, G Klebe, Klaus Reuter, Ralf FicnerAbstract:Abstract The crystal structure of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3α-HSDH) as well as the structure of its binary complex with NAD+ have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix αG and strand βG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3α-HSDH by the presence of a predominantly α-helical subdomain which is missing in all other SDRs of known structure.
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functional expression purification and characterization of 3alpha hydroxysteroid dehydrogenase carbonyl reductase from Comamonas testosteroni
Biochemical and Biophysical Research Communications, 2000Co-Authors: Edmund Maser, Eric Mobus, Guangming XiongAbstract: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.