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.
Highlights
Ketosteroid dehydrogenases are enzymes of biotechnological relevance that introduce a double bond into steroids as a first step toward their degradation
We show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with ⌬4-(5␣)-ketosteroid dehydrogenases (KSTDs) activity
We present the crystal structure of ⌬4-(5␣)-KSTD (Ro05698) from R. jostii RHA1 in its steroid-free and productbound form. These first structures of a KSTD enzyme allowed the identification of the active site residues involved in the dehydrogenation of 3-ketosteroid substrates and its reaction mechanism, which was confirmed through mutational analyses
Summary
Ketosteroid dehydrogenases are enzymes of biotechnological relevance that introduce a double bond into steroids as a first step toward their degradation. 3-Ketosteroid ⌬4-(5␣)-dehydrogenases (⌬4-(5␣)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5␣)-steroids. In the Rhodococcus jostii RHA1 genome [1], a single ⌬4kstD gene (ro05698) is present It codes for a protein of 490 amino acid residues, with an estimated molecular mass of 51.9 kDa. The gene has been cloned, and the enzyme has been overexpressed as a His6-tagged protein in Escherichia coli and purified [10]. We present the crystal structure of ⌬4-(5␣)-KSTD (Ro05698) from R. jostii RHA1 in its steroid-free and productbound form These first structures of a KSTD enzyme allowed the identification of the active site residues involved in the dehydrogenation of 3-ketosteroid substrates and its reaction mechanism, which was confirmed through mutational analyses
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