Structure and catalytic mechanism of 3-ketosteroid-Delta4-(5?)-dehydrogenase from Rhodococcus jostii RHA1 genome.

ABSTRACT: 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. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with ?4-(5?)-KSTD activity. The 1.6 ? 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 4?-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 5?-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by ?4-(5?)-KSTDs.

SUBMITTER: van Oosterwijk N 

PROVIDER: S-EPMC3438930 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature