Oligomerization and negative autoregulation of the LysR-type transcriptional regulator HsdR from Comamonas testosteroni

Gong, W. J., Xiong, G. M. and Maser, Edmund (2012) Oligomerization and negative autoregulation of the LysR-type transcriptional regulator HsdR from Comamonas testosteroni Journal of Steroid Biochemistry and Molecular Biology, 132 (3-5). pp. 203-211.

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Abstract

"3 alpha-Hydroxysteroid dehydrogenase/carbonyl reductase regulator" (HsdR) from Comamonas testosteroni (C. testosteroni) was identified as a member of the LysR-type transcriptional regulator (LTTR) family. We have shown previously that HsdR activates the expression of the hsdA gene, encoding 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR), which is an important enzyme involved in the degradation of steroid compounds. Phylogenetic analysis indicated that HsdR is related to the contact-regulated gene A (CrgA) from Neisseria meningitidis, which exists as a homooctamer. Therefore, to further elucidate the regulatory mechanism of HsdR, we investigated the oligomeric state and autoregulation of this transcriptional factor in the present study. To identify the active domains of HsdR, three truncated forms, HsdR Delta N (N-terminus deleted), HsdR Delta C (C-terminus deleted), and HsdR Delta NC (both N- and C-terminus deleted), were constructed and purified. 3 alpha-HSD/CR expression was measured by ELISA to detect the function of HsdR. Functional and biochemical analyses of wild type HsdR and its truncated forms indicated that HsdR may exist as an oligomer where the central domain is crucial for its oligomerization. Gel filtration chromatography revealed that there are two dominant oligomer forms which may be octamers and hexamers. According to electrophoretic mobility shift assays. HsdR specifically binds to its own promoter, where it negatively regulates its own expression. Therefore, the expression of non-functional HsdR variants (an hsdR-gfp fusion mutant and a hsdR gene disrupted mutant) increased compared to the wild type strain, because autorepression of HsdR was prevented. As a consequence, 3 alpha-HSD/CR expression in these hsdR mutant strains was impaired. Combined, in our study we provide evidence that the transcription factor HsdR is a component of the steroid degradation machinery in C. testosteroni, which is active as an oligomer and negatively regulates its own expression. (C) 2012 Elsevier Ltd. All rights reserved.

Document Type: Article
Additional Information: Univ Med Sch, Inst Toxicol & Pharmacol Nat Scientists, D-24105 Kiel, Germany. Maser, E (reprint author), Univ Med Sch, Inst Toxicol & Pharmacol Nat Scientists, Campus Kiel,Brunswiker Str 10, D-24105 Kiel, Germany. maser@toxi.uni-kiel.de
Keywords: Steroid degradation Comamonas testosteroni HsdR 3 alpha-HSD/CR Autorepression 3-alpha-hydroxysteroid dehydrogenase/carbonyl reductase neisseria-meningitidis pseudomonas-putida cross-linking expression protein family gene operon identification
Research affiliation: OceanRep > The Future Ocean - Cluster of Excellence
Kiel University
ISSN: 0960-0760
Projects: Future Ocean
Date Deposited: 14 May 2014 10:07
Last Modified: 14 May 2014 10:07
URI: http://eprints.uni-kiel.de/id/eprint/23995

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