Structure of alcohol oxidase from pichia pastoris by cryo-electron microscopy

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

A slice of the density near the fourfold surface.

<p>A salt bridge between Glu656 and Arg214 of different subunits (gold and light blue) may contribute to intersubunit contacts.</p

Peroxisomal AOX crystal packing.

<p>(a) Schematic representation of the peroxisomal AOX crystal packing (reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.ref014" target="_blank">14</a>] under a CC BY license, with permission from the American Society for Microbiology, original copyright 1992). AOX octamers are shown as square blocks with a cross indicating the fourfold axis. One unit cell of space group I432 with cell dimensions 228x228x228 Å is shown as a cube and contains 6 octamers (shaded grey). Each octamer has four neighbours, which are 90° rotated. This packing creates rows of octamers in three perpendicular directions, while the fourfold faces of six octamers line a hole (located in the center and at the eight vertices of the cube). (b) Class averages of a subset of picked particles containing a second molecule in the box. Classes showing a side view (1, 2, 4, 5, 9, 10, 11, 12) have a convex neighbour (top view) and classes showing a top view (3, 7, 8) a concave one (side view), suggesting a preferential 90° rotation between neighbours. (c) Part of a micrograph showing several AOX molecules arranged as in the crystals. Note the alternating arrangement of top view and side views. (d,e) Model of the crystal packing. The EM map was filtered to 10 Å for clarity. Neighbouring molecules are 90° rotated relative to each other and shifted 114 Å (half a unit cell). Molecules in the same orientation are shown in the same color. The yellow molecules are viewed along the fourfold axis. (d) shows one layer and (e) two layers. A unit cell is indicated in (e). (f, g) The crystal packing is mediated by helix H12. Two models are shown with each subunit in a different shade for clarity. In (f), the molecules are in the same orientation as in (d) and (e) and in (g) 45° rotated.</p

The active site.

<p>Domain colors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.g005" target="_blank">Fig 5a</a>. His567 and Asn616 play a role in catalysis. The side chains of the other indicated residues restrict substrate access to the active site and are probably involved in the specificity of AOX for small alcohols. His515 is part of a different subunit (grey).</p

Cryo-electron microscopy of AOX.

<p>(a) Micrograph of AOX collected on a JEOL 3200 FSC electron microscope using a K2 direct electron detector. The defocus was determined as 1100 nm. The scale bar represents 20 nm. (b) 2D class averages calculated using Relion show the octameric particle in different orientations.</p

Model of the AOX monomer.

<p>(a) Structural domains. The domain assignment for the GMC family [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.ref002" target="_blank">2</a>]) is as follows: FAD-binding domain (red); FAD covering lid (pink); extended FAD-binding domain (yellow); flavin attachment loop (green) and intermediate region (light green); substrate-binding domain (cyan). Helix H12, which forms crystal contacts, is shown in orange. The longest AOX-specific insert 481–548 is shown in dark blue, other AOX-specific sequences are colored purple. The FAD cofactor is blue. The location of the five β-sheets A-E and some α-helices (nomenclature from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.s001" target="_blank">S1 Fig</a>) and sequence mentioned in the text is indicated. The view of the monomer is along the fourfold axis from inside the complex. (b) Conservation of residues. Residues conserved in the GMC family (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.s001" target="_blank">S1 Fig</a>) are shown in red and residues conserved in AOX are green; unconserved residues are white. Structural elements unique for AOX are yellow. Note that the FAD binding domain (right; red in (a)) has high conservation in the GMC family, while the substrate binding domain centered around β-sheet C (left; cyan in (a)) is conserved between AOX species.</p