UBC Theses and Dissertations
The substrate specificity and conformational flexibility of ketosteroid hydroxylases Penfield, Jonathan
3-Ketosteroid-9α-hydroxylase (KshAB) is a Rieske oxygenase involved in bacterial steroid degradation. Bacteria such as Rhodococcus rhodochrous DSM43269 harbor up to five KshA homologues (numbered 1 to 5) that appear to be involved in degrading different steroids. Previous work indicated that KshA5 (DSM43269) transforms an unusually broad range of 3-ketosteroids while KshA1 (DSM43269) and KshAMtb of Mycobacterium tuberculosis have strong preference for 3-ketosteroids with side chains at C-17. To better understand KshAs in general, KshA1 and KshA5 were purified anaerobically and characterized. Steady-state kinetic studies revealed that KshA1 has 10- to 100-fold higher apparent specificity constant for ketosteroids possessing long C17 side chain such as 3-oxo-23,24-bisnorchola-1,4-dien-22-oate (4-BNC), and is thus similar to KshAMtb. By contrast, KshA5 had highest specificity for substrates with C17-oxo (e.g., apparent kcat/Km > 10⁵ s-¹ M-¹ for 4-estrendione and 5α-androstandione vs. 10² s-¹ M-¹ for 1,4-BNC-CoA). However, KshA5 displayed very strong substrate inhibition with 1,4-androstadiene-3,17-dione (ADD) and 4-BNC (KSS ~110 μM) despite hydroxylation well coupled to O₂ consumption and turnover occurring at reasonable rates (apparent kcat ~0.7 s-¹). Crystallographic structures of four KshA:substrate complexes were determined: KshA1:ADD (2.4 Å), KshAMtb:ADD (2.3 Å), KshA5:ADD (1.8 Å) and KshA5:1,4-BNC-CoA (2.6 Å). In each, the substrate was bound in a similar orientation with the steroid’s C9 closest to the active site iron. In comparison to a structure of substrate-free KshA5 (2.6 Å resolution), the catalytic iron was displaced up to 3.1 Å in the complexes with ADD and 1,4-BNC-CoA. This was accompanied by similar magnitude shift in the helices harboring the iron-coordinating residues. The net effect was an unusually large distance between the iron and C9 of the substrate (6.3 Å). Additionally, the active site opening of KshA5 was occluded from bulk solvent by a loop comprising residues 217 to 233 in substrate-free and ADD-bound structures while the KshA5:1,4-BNC-CoA complex exhibited an open active site, as observed in KshA1 and KshAMtb structures, containing a similar disordered loop region. The loop conformation in these structures and the ability of KshA5 to turnover CoA thioesters demonstrate unexpected conformational flexibility in correlation with interesting kinetic behavior in a Rieske oxygenase.
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