Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

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• 作者：Raina Boteva ; Antonie J. W. G. Visser ; Bruno Filippi ; Gert Vriend ; Marten Veenhuis ; and Ida J. van der Klei
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：April 20, 1999
• 年：1999
• 卷：38
• 期：16
• 页码：5034 - 5044
• 全文大小：223K
• 年卷期：v.38,no.16(April 20, 1999)
• ISSN：1520-4995

文摘

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidationin methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomeswhere the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AOactivation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris andthree putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitableintramolecular markers of the conformation and oligomeric state of AO. A direct relationship betweendissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescencelifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay componentwhich reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket.The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purifiedAO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structurewhich was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversionof -sheets into -helices. Our results are explained in relation to a 3D model of AO, which was builtbased on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger.The implications of our results for the current model of the in vivo AO assembly pathway are discussed.