4QDC

Crystal structure of 3-ketosteroid-9-alpha-hydroxylase 5 (KshA5) from R. rhodochrous in complex with FE2/S2 (INORGANIC) CLUSTER

Summary for 4QDC

• Entry DOI: 10.2210/pdb4qdc/pdb
• Related: 4QCK 4QDD 4QDF
• Descriptor: 3-ketosteroid 9alpha-hydroxylase oxygenase, FE2/S2 (INORGANIC) CLUSTER, FE (III) ION, ... (8 entities in total)
• Functional Keywords: mixed function oxygenases, oxidoreductase
• Biological source: Rhodococcus rhodochrous
• Total number of polymer chains: 1
• Total formula weight: 45197.50

Authors

Penfield, J.,Worrall, L.J.,Strynadka, N.C.,Eltis, L.D. (deposition date: 2014-05-13, release date: 2014-07-30, Last modification date: 2023-09-20)

Primary citation

Penfield, J.S.,Worrall, L.J.,Strynadka, N.C.,Eltis, L.D.
Substrate specificities and conformational flexibility of 3-ketosteroid 9 alpha-hydroxylases.
J.Biol.Chem., 289:25523-25536, 2014

PubMed Abstract: KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >10(5) s(-1) M(-1) for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (Ki S ∼100 μM). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue "mouth loop," which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.

PubMed: 25049233
DOI: 10.1074/jbc.M114.575886

Experimental method

X-RAY DIFFRACTION (1.9 Å)