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Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1)

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Abstract

In mammals, steroid hormones are synthesized from cholesterol that is metabolized by the mitochondrial CYP11A1 system leading to pregnenolone. The reduction equivalents for this reaction are provided by NADPH, via a small electron transfer chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). The reaction partners are involved in a series of transient interactions to realize the electron transfer from NADPH to CYP11A1. Here, we compared the ionic strength effect on the AdR/Adx and Adx/CYP11A1 interactions for wild-type Adx and mutant AdxS112W. Using surface plasmon resonance measurements, stopped flow kinetic investigations and analyses of the product formation, we were able to obtain new insights into the mechanism of these interactions. The replacement of serine 112 by tryptophan was demonstrated to lead to a dramatically decreased k off rate of the Adx/CYP11A1 complex, resulting in a four-fold decreased K d value and indicating a much higher stability of the complex involving the mutant. Stopped flow analysis at various ionic strengths and in different mixing modes revealed that the binding of reduced Adx to CYP11A1 seems to display the limiting step for electron transfer to CYP11A1 with pre-reduced AdxS112W being much more efficient than wild-type Adx. Finally, the dramatic increase in pregnenolone formation at higher ionic strength using the mutant demonstrates that the interaction of CYP11A1 with Adx is the rate-limiting step in substrate conversion and that hydrophobic interactions may considerably improve this interaction and the efficiency of product formation. The data are discussed using published structural data of the complexes.

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Abbreviations

AdR:

Recombinant wild-type adrenodoxin reductase

Adx:

Recombinant wild-type adrenodoxin

CYP:

Cytochrome P450

CYP11A1:

Cytochrom

P450scc:

Side chain cleavage enzyme from bovine adrenals

NADPH:

Nicotinamide adenine dinucleotide phosphate

FAD:

Flavine adenine dinucleotide

SPR:

Surface plasmon resonance

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Acknowledgments

The authors express their gratitude to Wolfgang Reinle for expert technical support and Dr. Susanne Zöllner for proofreading and supporting the realization of this manuscript.

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Authors and Affiliations

  1. Naturwissenschaftlich-Technische Fakultät III, Lehrstuhl für Biochemie, Universität des Saarlandes, Campus B2.2, 66123, Saarbrücken, Germany

    Burkhard Schiffler, Andy Zöllner & Rita Bernhardt

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  1. Burkhard Schiffler
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  2. Andy Zöllner
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  3. Rita Bernhardt
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Correspondence to Rita Bernhardt.

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Special Issue: Transient interactions in biology.

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Schiffler, B., Zöllner, A. & Bernhardt, R. Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1). Eur Biophys J 40, 1275–1282 (2011). https://doi.org/10.1007/s00249-011-0703-6

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  • DOI: https://doi.org/10.1007/s00249-011-0703-6

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