Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD.
The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo- and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.
Caldinelli
Laura
L
Department of Biotechnology and Molecular Sciences, University of Insubria, 21100 Varese, Italy.
Iametti
Stefania
S
Barbiroli
Alberto
A
Fessas
Dimitrios
D
Bonomi
Francesco
F
Piubelli
Luciano
L
Molla
Gianluca
G
Pollegioni
Loredano
L
D001051:Apoenzymes / Q000737:chemistry; D001951:Brevibacterium / Q000201:enzymology; D002151:Calorimetry; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D006639:Histidine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D002789:Cholesterol Oxidase