This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Florin, C. Articles by Plesiat, P. Search for Related Content PubMed PubMed Citation Articles by Florin, C. Articles by Plesiat, P.

 Previous Article  |  Next Article 

J. Bacteriol., Jun 1996, 3322-3330, Vol 178, No. 11
Copyright © 1996, American Society for Microbiology

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization

C Florin, T Kohler, M Grandguillot and P Plesiat
Laboratoire de Bacteriologie, Faculte de Medecine, Besancon, France.

Comamonas testosteroni delta 4(5 alpha)- and delta1-dehydrogenases [delta4(5alpha)- and delta1DH] are key enzymes in the degradation of steroids having an A:B ring fusion in a trans configuration. We previously reported the isolation of the delta1dh gene (P. Plesiat, M. Grandguillot, S. Harayama, S. Vragar, and Y. Michel Briand, J. Bacteriol. 173:7219-7227, 1991). In this study, the gene encoding delta 4(5 alpha)DH was cloned in Escherichia coli on a 16-kbp BamHI fragment by screening a genomic bank of C. testosteroni ATCC 17410 with a probe derived from delta1dh. Subcloning experiments in plasmid pUC19 mapped delta 4(5 alpha)dh immediately downstream of delta1dh. The enzyme was overexpressed 18-fold in cells of E. coli JM109 carrying a 2.5-kbp cloned fragment (plasmid pXE25). However, much higher levels of enzymatic activity (264-fold) were obtained in Pseudomonas putida KT2440, using pMMB208 as an expression vector. Studies with crude lysates of KT2440 showed that delta4(5alpha)DH exhibits higher specificity and higher activity toward delta l-androstene-3,17-dione than toward the saturated derivative 5 alpha-androstane-3,17-dione. The reaction was found to be irreversible and to use efficiently typical flavoprotein electron acceptors; optimal conditions for the enzyme activity were pH 8 and 40 degrees C. Analysis of the nucleotide sequence of the insert of pXE25 revealed an open reading frame of 1,593 bp preceded by a putative ribosome-binding site and followed by a potential transcription terminator. The amino acid sequence of the deduced peptide showed a typical flavin adenine dinucleotide-binding site in its N-terminal region, confirming the flavoproteinic structure of delta 4(5 alpha)DH. The predicted molecular mass was consistent with that of the enzyme expressed in a T7 polymerase system (60 kDa). Alignment between delta 4(5 alpha)dh and delta1dh indicated that both genes, though coding for functionally related enzymes, do not derive from a common ancestor.

This article has been cited by other articles:

• Gohler, A., Xiong, G., Paulsen, S., Trentmann, G., Maser, E. (2008). Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni. J. Biol. Chem. 283: 17380-17390 [Abstract] [Full Text]  
• Chiang, Y.-R., Ismail, W., Gallien, S., Heintz, D., Van Dorsselaer, A., Fuchs, G. (2008). Cholest-4-En-3-One-{Delta}1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism. Appl. Environ. Microbiol. 74: 107-113 [Abstract] [Full Text]  
• Birkenmaier, A., Holert, J., Erdbrink, H., Moeller, H. M., Friemel, A., Schoenenberger, R., Suter, M. J.-F., Klebensberger, J., Philipp, B. (2007). Biochemical and Genetic Investigation of Initial Reactions in Aerobic Degradation of the Bile Acid Cholate in Pseudomonas sp. Strain Chol1. J. Bacteriol. 189: 7165-7173 [Abstract] [Full Text]  
• Brzostek, A., Sliwinski, T., Rumijowska-Galewicz, A., Korycka-Machala, M., Dziadek, J. (2005). Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis. Microbiology 151: 2393-2402 [Abstract] [Full Text]  
• Pruneda-Paz, J. L., Linares, M., Cabrera, J. E., Genti-Raimondi, S. (2004). TeiR, a LuxR-Type Transcription Factor Required for Testosterone Degradation in Comamonas testosteroni. J. Bacteriol. 186: 1430-1437 [Abstract] [Full Text]  
• Xiong, G., Martin, H.-J., Maser, E. (2003). Identification and Characterization of a Novel Translational Repressor of the Steroid-inducible 3{alpha}-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni. J. Biol. Chem. 278: 47400-47407 [Abstract] [Full Text]  
• Horinouchi, M., Hayashi, T., Yamamoto, T., Kudo, T. (2003). 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. Appl. Environ. Microbiol. 69: 4421-4430 [Abstract] [Full Text]  
• Horinouchi, M., Hayashi, T., Koshino, H., Yamamoto, T., Kudo, T. (2003). Gene Encoding the Hydrolase for the Product of the meta-Cleavage Reaction in Testosterone Degradation by Comamonas testosteroni. Appl. Environ. Microbiol. 69: 2139-2152 [Abstract] [Full Text]  
• Horinouchi, M., Yamamoto, T., Taguchi, K., Arai, H., Kudo, T. (2001). Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441. Microbiology 147: 3367-3375 [Abstract] [Full Text]  
• van der Geize, R., Hessels, G. I., van Gerwen, R., Vrijbloed, J. W., van der Meijden, P., Dijkhuizen, L. (2000). Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Delta 1-Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1. Appl. Environ. Microbiol. 66: 2029-2036 [Abstract] [Full Text]  
• Xiong, G., Maser, E. (2001). Regulation of the Steroid-inducible 3alpha -Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni. J. Biol. Chem. 276: 9961-9970 [Abstract] [Full Text]