Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis
| Autor: | Lindsay D. Eltis, Natalie C. J. Strynadka, Igor D'Angelo, Jenna K. Capyk |
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| Rok vydání: | 2009 |
| Předmět: |
Oxygenase
Stereochemistry Protein subunit Molecular Conformation Reductase Crystallography X-Ray Ligands Biochemistry Mixed Function Oxygenases Electron Transport Complex III chemistry.chemical_compound Bacterial Proteins Oxidoreductase Ketosteroid Enzyme kinetics Molecular Biology chemistry.chemical_classification Dose-Response Relationship Drug biology Chemistry Temperature Active site Mycobacterium tuberculosis Cell Biology Hydrogen-Ion Concentration Protein Structure Tertiary Kinetics Cholesterol Enzyme Models Chemical Protein Structure and Folding Oxygenases biology.protein Dimerization Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 284:9937-9946 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m900719200 |
| Popis: | KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance. |
| Databáze: | OpenAIRE |
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