title: Structure and catalytic mechanism of 3-ketosteroid-Delta4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 genome.
abstract: 3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.
authors: Laboratory of Biophysical Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands.
authors: Niels
authors: N
authors: van Oosterwijk
authors: Jan
authors: J
authors: Knol
authors: Lubbert
authors: L
authors: Dijkhuizen
authors: Robert
authors: R
authors: van der Geize
authors: Bauke W
authors: BW
authors: Dijkstra
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D016680:Genome, Bacterial; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases