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.
Highlights
Contrary to most eukaryotic organisms, several yeast species can utilize methanol as sole carbon and energy source, enabling such methylotrophic yeasts to occupy an ecological niche [1]
An enzymatic activity assay with purified P. pastoris AOX1 revealed that the enzyme is active at pH 7.4 and 30°C exhibiting a KM value of 0.6 mM and a kcat of 343 min-1 for the substrate methanol under these conditions (S1 Table)
These kinetic parameters are in reasonable agreement with previously reported values (Zhang et al reported a KM of 0.6–1.0 mM and a kcat of 136–270 min-1 for Crystal Structure of AOX1 from Pichia pastoris conversion of methanol at 30°C and pH 7.5) [24]
Summary
Contrary to most eukaryotic organisms, several yeast species can utilize methanol as sole carbon and energy source, enabling such methylotrophic yeasts to occupy an ecological niche [1]. The methanol assimilation pathway is initiated by the oxidation of that alcohol to formaldehyde. This reaction is catalyzed by the FAD-dependent alcohol oxidase (AOX; EC 1.1.3.13), which belongs to the family of glucose-methanol-choline (GMC) oxidoreductases [2]. This enzyme oxidizes in vitro other short aliphatic alcohols such as ethanol and 1-propanol, what gave its generalized name alcohol oxidase. The AOX gene is subject to a strong carbon catabolite repression [3, 4]. Its synthesis is strictly regulated by induction and repression/derepression mechanisms that occur at the transcriptional level with identified regulating components being a hexose transporter [5], a glucokinase and a PLOS ONE | DOI:10.1371/journal.pone.0149846 February 23, 2016
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