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Substrate specificities and conformational flexibility of 3-ketosteroid 9α-hydroxylases.
Penfield, Jonathan S; Worrall, Liam J; Strynadka, Natalie C; Eltis, Lindsay D.
Afiliação
  • Penfield JS; From the Departments of Biochemistry and Molecular Biology and.
  • Worrall LJ; From the Departments of Biochemistry and Molecular Biology and.
  • Strynadka NC; From the Departments of Biochemistry and Molecular Biology and.
  • Eltis LD; 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.
J Biol Chem ; 289(37): 25523-36, 2014 Sep 12.
Article em En | MEDLINE | ID: mdl-25049233
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.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Relação Estrutura-Atividade / Proteínas de Bactérias / Rhodococcus / Colesterol / Oxigenases de Função Mista Idioma: En Revista: J Biol Chem Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Relação Estrutura-Atividade / Proteínas de Bactérias / Rhodococcus / Colesterol / Oxigenases de Função Mista Idioma: En Revista: J Biol Chem Ano de publicação: 2014 Tipo de documento: Article