title: Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism.
abstract: 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.
authors.affiliation: Mikrobiologie, Institut Biologie II, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany.
authors.forename: Yin-Ru
authors.initials: YR
authors.lastname: Chiang
authors.forename: Wael
authors.initials: W
authors.lastname: Ismail
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gallien
authors.forename: Dimitri
authors.initials: D
authors.lastname: Heintz
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Georg
authors.initials: G
authors.lastname: Fuchs
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002783:Cholestenones / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D003345:Corticosterone / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004970:Estrone / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008751:Methylene Blue / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D043362:Rhodocyclaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D002783:Cholestenones; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D009566:Nitrates; D011994:Recombinant Proteins; C526532:cholesta-1,4-dien-3-one; D004970:Estrone; D011374:Progesterone; C000475:cholest-4-en-3-one; D002784:Cholesterol; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases; D008751:Methylene Blue; D003345:Corticosterone