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Applied and Environmental Microbiology, January 2008, p. 107-113, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01968-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cholest-4-En-3-One-{Delta}1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism{triangledown}

Yin-Ru Chiang,1 Wael Ismail,1 Sébastien Gallien,2 Dimitri Heintz,2 Alain Van Dorsselaer,2 and Georg Fuchs1*

Mikrobiologie, Institut Biologie II, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany,1 Laboratoire de Spéctrometrie de Masse Bio-Organique, CRNS, ECPM, Université Louis Pasteur, Strasbourg, France2

Received 28 August 2007/ Accepted 31 October 2007

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-{Delta}1-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected {Delta}1-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-{Delta}1-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.

* Corresponding author. Mailing address: Mikrobiologie, Institut Biologie II, Schänzlestr. 1, D-79104 Freiburg, Germany. Phone: (49) 761-2032649. Fax: (49) 761-2032626. E-mail: georg.fuchs{at}biologie.uni-freiburg.de

{triangledown} Published ahead of print on 9 November 2007.

Applied and Environmental Microbiology, January 2008, p. 107-113, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01968-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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