Abstract
The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.
Highlights
Methylotrophic yeasts can utilize methanol as sole carbon source
The first step of methanol metabolism, the oxidation with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX) (EC 1.1.3.13) and occurs in specialized organelles, the peroxisomes, where H2O2 is decomposed by catalase
AOX catalyses the oxidation of small primary alcohols [4] using a two-step mechanism, first a reductive half-reaction, where the substrate is oxidised while the FAD is reduced to FADH2, followed by an oxidative half-reaction, where the flavin is reoxidized by O2 and H2O2 is produced [5, 6]
Summary
Methylotrophic yeasts can utilize methanol as sole carbon source. The first step of methanol metabolism, the oxidation with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX) (EC 1.1.3.13) and occurs in specialized organelles, the peroxisomes, where H2O2 is decomposed by catalase. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities.
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