Crystal Structure of Alcohol Oxidase from Pichia pastoris

Abstract

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring.

Fig 1. The structure of one P. pastoris AOX1 subunit.

The two views are related by rotating the molecule by 180 degrees. (a) Rainbow colored cartoon representation of the AOX1 monomer. FAD is depicted in ball-and-stick representation; the position of the PP loop (residues 13–17) is highlighted as blue balls and sticks. N- and C-termini are labeled. (b) Superposition with Choline oxidase (PDB: 3LJP) and domain organization of the AOX1 subunit, both proteins represented as cartoon. The FAD-binding domain (AOX1, residues 1–155, 192–306 and 568–663) is colored wheat and a substrate-binding domain (AOX1, residues 156–191 and 307–567) is colored dirty violet. Residues forming insertions are colored purple and red for substrate- and FAD-binding domains, respectively. Choline oxidase is colored grey. (c) Monomer of AOX1 with residues involved in octamer formation depicted as surface representation, colored as in b (d) Monomer of AOX1 with residues involved in dimer formation (inter tetramer interactions) highlighted with yellow dots. Colors are chosen as in (b) and (c).

Fig 2. The quaternary structure of P. pastoris AOX1.

Top view represents an octamer with individually colored monomers in surface representation, of which one monomer is colored as in Fig 1C. The middle view represents the cartoon representation of the octamer with one inter-tetramer dimer highlighted by different coloring: one monomer as in Fig 1D and the second one with substrate binding domain colored in dark olive and insertions in marine. Zoomed in view of the marked rectangular area depicting more detailed intersubunit interactions is presented beneath the individual octamer orientation. (a) View along 2-fold axis (b) AOX1 octamer upon rotation the “a” view by 90 degrees. (c) AOX1 octamer viewed along the 4-fold axis upon rotation the “b” view by 90 degrees. The second tetramer is depicted as surface.

Fig 3. Modified a-FAD.

Calculated mFo-DFc omit electron density map contoured at 3.5 σ level clearly indicates the presence of modified a-FAD molecule bound to the active site of AOX1 monomer. The bound cofactor molecule is presented in ball-and-stick representation and the accompanying water molecule is depicted as a red sphere. Atoms of the isoalloxazine ring that are important for catalysis are marked (N10, N5) as well as carbon (C2’) and oxygen (O2’) atoms affected by epimerization. (a) Not modified FAD cofactor. (b) Modified a-FAD, the changed chiral configuration center is labeled as C2’.

Fig 4. Comparison of AOX1 and choline oxidase active sites.

AOX1 monomer is colored as in Fig 2C, the CO is colored grey. The residues delimiting the substrate binding cavity are labeled and represented as sticks. Modified a-FAD is depicted in ball-and-stick representation. (a) The active site of AOX1 with cavities depicted in atom colored surface representation. The substrate binding cavity is marked with blurred red rim. The water molecule, which is bound close to the isoalloxazine ring, is depicted as sphere. Polar interactions are marked as dashed lines. (b) The active site of Choline oxidase with cavities depicted in surface representation. The substrate binding cavity is marked with blurred red rim. (c) Cartoon representation of superimposed AOX1 and Choline Oxidase monomers.

Fig 5. Active site comparison between AOX1 and choline oxidase complexed with glycine betaine.

AOX1 monomer is colored as in Fig 2C and Fig 4, the CO is colored grey. Residues delimiting the substrate binding cavity are labeled and represented as sticks. Modified a-FAD is depicted in ball-and-stick representation. Superposition with the closely related CO in a complex with product glycine betaine (PDB id: 4MJW) indicates that the water molecule in the AOX1 active site, depicted as magenta sphere, occupies similar position as one oxygen of the carboxylic group of the bound product molecule.