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Cholesterol oxidase from Brevibacterium sterolicum : the relationship between covalent flavinylation and redox properties

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2001

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Motteran, Laura
Pilone, Mirella S.
Molla, Gianluca
Pollegioni, Loredano

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Cholesterol Oxidase
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Open Access Green
Core Facility der Universität Konstanz

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Journal of Biological Chemistry. 2001, 276(21), pp. 18024-18030. ISSN 0021-9258. eISSN 1083-351X. Available under: doi: 10.1074/jbc.M010953200

Zusammenfassung

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His69 of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8α-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.

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570 Biowissenschaften, Biologie

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ISO 690MOTTERAN, Laura, Mirella S. PILONE, Gianluca MOLLA, Sandro GHISLA, Loredano POLLEGIONI, 2001. Cholesterol oxidase from Brevibacterium sterolicum : the relationship between covalent flavinylation and redox properties. In: Journal of Biological Chemistry. 2001, 276(21), pp. 18024-18030. ISSN 0021-9258. eISSN 1083-351X. Available under: doi: 10.1074/jbc.M010953200
BibTex
@article{Motteran2001Chole-6857,
  year={2001},
  doi={10.1074/jbc.M010953200},
  title={Cholesterol oxidase from Brevibacterium sterolicum : the relationship between covalent flavinylation and redox properties},
  number={21},
  volume={276},
  issn={0021-9258},
  journal={Journal of Biological Chemistry},
  pages={18024--18030},
  author={Motteran, Laura and Pilone, Mirella S. and Molla, Gianluca and Ghisla, Sandro and Pollegioni, Loredano}
}
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