Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

Lim, Louis; Molla, Gianluca; Guinn, Nicole; Ghisla, Sandro; Pollegioni, Loredano; Vrielink, Alice

Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

Dateien

Structural_and_kinetic_analyses_of_the_H121A_mutant_of_cholesterol_oxidase.pdfGröße: 717.99 KBDownloads: 386

Datum

2006

Autor:innen

Lim, Louis

Molla, Gianluca

Guinn, Nicole

Ghisla, Sandro

Pollegioni, Loredano

Vrielink, Alice

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Biochemical Journal. 2006, 400(1), pp. 13-22. ISSN 0264-6021. eISSN 1470-8728. Available under: doi: 10.1042/BJ20060664

Zusammenfassung

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a ≈100 mV decrease in the midpoint redox potential and a ≈40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024 18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 Å resolution (1 Å≡0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180° about χ1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

covalent flavin, flavoprotein, kinetics, protein structure, redox, structure function relationships

Zitieren

ISO 690

LIM, Louis, Gianluca MOLLA, Nicole GUINN, Sandro GHISLA, Loredano POLLEGIONI, Alice VRIELINK, 2006. Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. In: Biochemical Journal. 2006, 400(1), pp. 13-22. ISSN 0264-6021. eISSN 1470-8728. Available under: doi: 10.1042/BJ20060664

BibTex

@article{Lim2006Struc-7776,
  year={2006},
  doi={10.1042/BJ20060664},
  title={Structural and kinetic analyses of the H121A mutant of cholesterol oxidase},
  number={1},
  volume={400},
  issn={0264-6021},
  journal={Biochemical Journal},
  pages={13--22},
  author={Lim, Louis and Molla, Gianluca and Guinn, Nicole and Ghisla, Sandro and Pollegioni, Loredano and Vrielink, Alice}
}

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Universitätsbibliographie

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