Substrate specificities and conformational flexibility of 3-Ketosteroid 9{alpha}-hydroxylases [Enzymology]

July 21st, 2014 by Penfield, J. S., Worrall, L. J., Strynadka, N. C., Eltis, L. D.

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 highest apparent specificity (kcat/KM) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholestra-1.4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had highest apparent specificity for substrates with no C17 side chain (kcat/KM > 105 s-1M-1 for 4-estrendione, 5α-andro-standione and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ~100 μM). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had 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 orientated towards the active site opening. Comparisons suggest Tyr245 and Phe297 are determinants of KshA1's 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 to substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochromes P450.