This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Petrusma, M.
Right arrow Articles by van der Geize, R.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petrusma, M.
Right arrow Articles by van der Geize, R.
Agricola
Right arrow Articles by Petrusma, M.
Right arrow Articles by van der Geize, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2009, p. 5300-5307, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00066-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9{alpha}-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity{triangledown} ,{dagger}

M. Petrusma, L. Dijkhuizen,* and R. van der Geize

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands

Received 12 January 2009/ Accepted 19 June 2009

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9{alpha}-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9{alpha}-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5{alpha}-H, 5β-H, {Delta}1, and {Delta}4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid {Delta}1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.

* Corresponding author. Mailing address: Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31 (50) 3632153. Fax: 31 (50) 3632154. E-mail: L.Dijkhuizen{at}rug.nl

{triangledown} Published ahead of print on 26 June 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2009, p. 5300-5307, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00066-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»