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J Biol Chem. 2014 Sep 12;289(37):25523-36. doi: 10.1074/jbc.M114.575886. Epub 2014 Jul 21.

Substrate specificities and conformational flexibility of 3-ketosteroid 9α-hydroxylases.

Author information

1
From the Departments of Biochemistry and Molecular Biology and.
2
From the Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada leltis@mail.ubc.ca.

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.

KEYWORDS:

Actinobacteria; Bacterial Metabolism; Bile Acid; Cholesterol; Metalloenzyme; Mycobacterium tuberculosis; Oxygenase

PMID:
25049233
PMCID:
PMC4162158
DOI:
10.1074/jbc.M114.575886
[Indexed for MEDLINE]
Free PMC Article

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