title: Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin.
abstract: 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 His(69) 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 (approximately 3 versus approximately 5 M, respectively), and the mutant protein had a melting temperature approximately 10-15 degrees 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.
authors.affiliation: Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.forename: Alberto
authors.initials: A
authors.lastname: Barbiroli
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fessas
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Mirella S
authors.initials: MS
authors.lastname: Pilone
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000494:pharmacology; D001951:Brevibacterium / Q000201:enzymology*; D002151:Calorimetry; D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D002789:Cholesterol Oxidase / Q000737:chemistry*; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005231:Fatty Acids, Unsaturated; D005415:Flavins / Q000737:chemistry*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays; D014508:Urea / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000817:Anilino Naphthalenesulfonates; D018076:DNA, Complementary; D005231:Fatty Acids, Unsaturated; D005415:Flavins; C055649:9-(4-chlorophenyl) 7,7-dimethyl-5,8-nonadienoic acid methyl ester; C027132:1-anilino-8-naphthalenesulfonate; D002248:Carbon Monoxide; D014364:Tryptophan; D014508:Urea; D006861:Hydrogen Peroxide; D002789:Cholesterol Oxidase