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

Abstract

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which 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 purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based 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.

Octameric alcohol oxidase dissociates into stable, soluble monomers upon incubation with dimethylsulfoxide

“…While octameric AO is estimated [15] to contain about 27% a-helical and 44% b-sheet structure (representing a total of about 71% regular secondary structure), monomeric AO is estimated to contain only about 18% a-helical and 30% b-sheet structure (in total 48% regular secondary structure). Thus, the estimated secondary structure content of octameric AO-but not of monomeric AO-is very similar to that of the previously described homology model [8], which contains 30.1% helical structure and 41.7% b-structure, as determined by WHATIF [16]. Finally, we determined the melting temperatures (T m ) of native, octameric AO and DMSO-treated monomeric AO (data not shown).…”

“…AO monomers contain 9 Trp and 31 Tyr residues, but the fluorescence emission spectrum of native AO is dominated by Trp fluorescence upon excitation at 295 nm (Fig. 2) [8]. DMSO treatment resulted in a shift of the emission maximum to the longer wavelength amounting 5 nm upon incubation in 50% DMSO.…”

“…FAD bound to AO mainly acts as an acceptor of tryptophan fluorescence because of the significant overlap between its second electronic absorption band and the tryptophan fluorescence spectrum [8]. Increasing DMSO concentrations resulted in enhanced Trp and FAD fluorescence ( Fig.…”

“…Incubation of native AO in 80% glycerol resulted in dissociation of the octamers into the constituting subunits. However, FAD remained bound to these subunits [7,8]. Moreover, the presence of a highly viscous solvent overshadows the results of studies of the dynamic properties of proteins and is likely to exert unwanted side effects on HpPyc1p in reconstitution experiments.…”

Deflavination of flavo-oxidases by nucleophilic reagents

“…The mechanism behind this phenomenon, however, remained obscure. Spectroscopic characterization of both octameric and monomeric H. polymorpha and Pichia pastoris AO showed signi¢cant conformational perturbations of the molecules upon FAD release thus demonstrating the importance of this co-factor for the structural integrity of the enzymatically active conformations of the two enzymes [14].…”