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. Published by Cold Spring Harbor Laboratory Press. Copyright

Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD / L. Caldinelli, S. Iametti, A. Barbiroli, D. Fessas, F. Bonomi, L. Piubelli, G. Molla, L. Pollegioni. - In: PROTEIN SCIENCE. - ISSN 0961-8368. - 17:3(2008), pp. 409-419.

Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD

S. Iametti
Secondo
;
A. Barbiroli;D. Fessas;F. Bonomi;
2008

Abstract

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. Published by Cold Spring Harbor Laboratory Press. Copyright
Cofactor binding; Conformational change; Flavoprotein; Fluorescence; Mutagenesis
Settore BIO/10 - Biochimica
Settore CHIM/02 - Chimica Fisica
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/57222
Citazioni
  • ???jsp.display-item.citation.pmc??? 4
  • Scopus 19
  • ???jsp.display-item.citation.isi??? 19
social impact