Enzymes for Research, Diagnostic and Industrial Use

Alcohol Oxidase

Official Full Name
Alcohol Oxidase
In enzymology, an alcohol oxidase (EC is an enzyme that catalyzes the chemical reaction a primary alcohol + O2 ↔ an aldehyde + H2O2. Thus, the two substrates of this enzyme are primary alcohol and O2, whereas its two products are aldehyde and H2O2. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor.
ethanol oxidase; alcohol oxidase; EC; Methanol oxidase; 9073-63-6

Product Name
EC No.
ProductNamealcohol oxidase
CAS No.9073-63-6
CAS No.9073-63-6
SourceHansenula sp.
CAS No.9073-63-6
SourcePichia pastoris
CAS No.9073-63-6
SourceCandida sp.
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Alcohol oxidase (AOX, EC is a flavoenzyme, an oxidoreductase that catalyzes the oxidation of alcohol to the corresponding carbonyl compound and simultaneously releases hydrogen peroxide. The systematic name of this enzyme is alcohol: oxygen oxidoreductase. Other names in common use include ethanol oxidase, methanol oxidase. According to substrate specificity, alcohol oxidases can be broadly divided into four different groups, namely short-chain alcohol oxidase (SCAO), long-chain alcohol oxidase (LCAO), aromatic alcohol oxidase (AAO), and secondary alcohol oxidase (SAO). Alcohol oxidases are mainly derived from bacteria, yeast, fungi, plants, insects and mollusks. SCAO and LCAO are mainly intracellular, AAO and SAO are mostly secreted into the medium.


AOX requires a flavin-based cofactor to catalyze the conversion of alcohols to the corresponding aldehydes or ketones. The FAD in AOX is closely related to the redox center of the enzyme and participates in the transfer of hydride ions from the alcohol substrate to molecular oxygen, thereby producing H2O2. The activity of AOX was originally reported in Polystictus versicolor, and later the researchers isolated an enzyme from the basidiomycete that catalyzes the oxidation of methanol and other lower alcohols to the corresponding aldehydes and H2O2. They then named the enzyme "alcohol oxidase", purified it to a crystalline state, and found that FAD is a prosthetic group of the enzyme. According to substrate specificity, alcohol oxidase can be broadly divided into four different groups, namely short chain alcohol oxidase (SCAO), long chain alcohol oxidase (LCAO), aromatic alcohol oxidase (AAO) and secondary alcohol oxidase. (SAO).


Like other members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, Pichia pastoris AOX1 contains two characteristic domains, including the FAD binding domain (residues 1-155, 192-306, and 568-663) and the substrate binding domain (residues 156-191 and 307-567). The most conserved region is the FAD binding domain, which contains four sequence fragments distributed throughout the primary sequence, with the characteristic FAD nucleotide binding site sequences GXGXXG, and GGGSSG in P. pastoris AOX1.

The structure of one <em>P. pastoris</em> AOX1 subunit. Figure 1. The structure of one P. pastoris AOX1 subunit. (Koch C. 2016)

The mature active form of AOX1 is an oligomer comprising eight identical subunits, each of which carries a non-covalently bound flavin adenine dinucleotide (FAD) molecule as a prosthetic group. The P. pastoris AOX1 crystal is a homo-octamer with a size of about 121.8 Å x 133.8 Å x 134.5 Å. The octamer appears to be a tetramer of dimers, but can also be described as two tetramers placed face to face. The two tetramers interact only through the substrate binding domains, while the FAD binding domains occupy the outer surface of the oligomer. In the tetramer, each monomer forms 20 hydrogen bonds and 8 salt bridges with each of two adjacent subunits, and occupies 7% (1985 Å2) of the contact surface area in pairs interaction. In contrast, each inter-tetramer dimer is stabilized by a wider inter-subunit contacts (40 hydrogen bonds), burying 10% (2700 Å2) of contact surface area. When an octamer is formed, each monomer forms about 115 hydrogen bonds and 16 salt bridges with adjacent subunits and buries about 29% (8300 Å2) of its contact surface area, which also indicates the strength of the octamer. The formation of the AOX1 oligomeric state is primarily facilitated by the insertion of numerous interactions between the monomers (residues 326-351, 486-548, 247-251, 638-663). Although one insertion is located in the substrate binding domain (367-386), a helix-loop-helix motif is formed that is located away from the other monomers and therefore does not participate in any inter-subunit interaction, the remaining two insertions ( 326-351, 486-548) are involved in the formation of tetramer and dimer subassemblies of AOX1 octamer. Their length and inherent structural plasticity combined with the face-to-face orientation of the individual facilitates the creation of a wide range of interaction networks in which each plug-in simultaneously participates in the stabilization of the two sub-assemblies. In contrast, the distant short loop extension (residues 247-251) and the C-terminal extension of the FAD binding domain participate in the formation of tetramers only by the interaction of two adjacent monomers involved in the formation of the tetramer.

The quaternary structure of P. pastoris Figure 2. The quaternary structure of P. pastoris AOX1. (Koch C. 2016)

Catalytic Mechanism

Analysis of the crystal structure of AOX1 showed that the active site was inaccessible to the solvent. Although the AOX1 catalytic center is solvent inaccessible, electron density peaks corresponding to water molecules can be observed at all active sites. The water molecule occupies a small cavity with a volume of about 135 Å3 and it is hydrogen bonded to the side chain of Asn616, His567 and the N5 atom of the isoalloxasine ring. Further studies have found that the side chains of Phe98 and Met100 may be structural elements that allow the substrate to enter the active site. The catalytic center of the eight subunits of AOX1 each contains a non-covalently bound FAD molecule.

The active site of AOX1. Figure 3. The active site of AOX1. (Koch C. 2016)

Methylotrophic yeast has evolved a regulatory mechanism to fine tune the activity of alcohol oxidase by automatically converting naturally occurring ribityl FAD moieties into modified arabityl FAD (a-FAD). a-FAD slightly reduces Vmax and significantly reduces the Km value of the enzyme for substrate methanol, which is physiologically relevant in cultures of low methanol concentrations. Since the AOX-catalyzed multi-step reaction is directly dependent on the redox potential of the bound cofactor, an increase in the FAD redox potential will contribute to the overall conversion of the enzyme. A change in chiral configuration at atomic C2' in the FAD of AOX results in the formation of new intramolecular hydrogen bond between the modified a-FAD aryl C2' OH group and the N1 atom of the isoalloxasine ring system, most likely modulates the redox potential of the FAD cofactor. The catalytic oxidation reaction begins by removing the substrate hydroxyl proton by catalytic base and forming an alkoxide species before the hydride transfer from the substrate α-carbon. For other alcohol oxidases (AAO, GO, CDH, P2O, PNO), the conservative His of His/His, His/Asn or His/Pro pairs is considered to be a catalytic base and therefore carrying a partial positive charge during the catalysis. His567 in AOX1 was identified as a catalytic base that activates alcohol. The stepwise reaction catalyzed by AOX1 requires the enzyme to stabilize the negative charge of the alkoxide by intermolecular interaction, which may be formed by two conserved polar residues, His567 and Asn 616. This residue is located at a hydrogen bond remote from the alkoxide site. Since the transfer of the hydride ion is carried out through the N5 position of the isoalloxasine ring, the polar group adjacent to the N5 atom can stabilize the transition state. In AOX1, the residue in which the OH group is placed is Phe98.

Modified a-FAD. Figure 4. Modified a-FAD. (Koch C. 2016)


  1. Goswami, P., Chinnadayyala, S.S., Chakraborty, M., Kumar, A.K., Kakoti, A. An overview on alcohol oxidases and their potential applications. Appl Microbiol Biotechnol, 2013, 97(10):4259-75.
  2. Koch, C., Neumann, P., Valerius, O., Feussner, I., Ficner, R. Crystal structure of alcohol oxidase from Pichia pastoris. PLoS One, 2016, 11(2): e0149846.

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