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J. Biol. Chem., Vol. 280, Issue 24, 22572-22581, June 17, 2005

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Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin^*

Laura Caldinelli, Stefania Iametti, Alberto Barbiroli, Francesco Bonomi, Dimitrios Fessas¶, Gianluca Molla, Mirella S. Pilone, and Loredano Pollegioni||

From the Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy and DISMA and ^¶DISTAM, University of Milan, via Celoria 2, 20133 Milan, Italy

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His⁶⁹ residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (3 versus 5 M, respectively), and the mutant protein had a melting temperature 10–15 °C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.

Received for publication, January 18, 2005 , and in revised form, March 17, 2005.

^* This work was supported by grants from Italian Ministero dell' Istruzione, dell' Università e della Ricerca (Prot 2004058243), Fondo di Ateneo per la Ricerca 2004 (to M. S. P. and L. P.), and Fondazione CARIPLO (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy. Tel.: 0332-421506; Fax: 0332-421500; E-mail: loredano.pollegioni{at}uninsubria.it.

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