title: A single loop is essential for the octamerization of vanillyl alcohol oxidase.
abstract: The VAO/PCMH family of flavoenzymes is a family of structurally related proteins that catalyse a wide range of oxidation reactions. It contains a subfamily of enzymes that catalyse the oxidation of para-substituted phenols using covalently bound FAD cofactors (the 4PO subfamily). This subfamily is composed of two oxidases, vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO), and two flavocytochrome dehydrogenases, para-cresol methylhydroxylase (PCMH) and eugenol hydroxylase (EUGH). Although they catalyse similar reactions, these enzymes differ in terms of their electron acceptor preference and oligomerization state. For example, VAO forms homo-octamers that can be described as tetramers of stable dimers, whereas EUGO is exclusively dimeric in solution. A possible explanation for this difference is the presence of a loop at the dimer-dimer interface in VAO that is not present in EUGO. Here, the role played by this loop in determining the quaternary structure of these enzymes is investigated. A VAO variant where the loop was deleted, loopless VAO, exclusively formed dimers. However, introduction of the loop into EUGO was not sufficient to induce its octamerization. Neither variant displayed major changes in its catalytic properties as compared to the wild-type enzyme. Bioinformatic analysis revealed that the presence of the loop is conserved within putative fungal oxidases of the 4PO subgroup, but it is never found in putative bacterial oxidases or dehydrogenases. Our results shed light on the molecular mechanism of homo-oligomerization of VAO and the importance of oligomerization for its enzymatic function.
  
  
  
  p-cresol methylhydroxylase (4-methylphenol:acceptor oxidoreductase (methyl-hydroxylating), EC 1.17.99.1); vanillyl alcohol oxidase (vanillyl alcohol:oxygen oxidoreductase, EC 1.1.3.38).
authors.affiliation: Laboratory of Biochemistry, Wageningen University, The Netherlands.
authors.forename: Tom A
authors.initials: TA
authors.lastname: Ewing
authors.affiliation: Laboratory of Biochemistry, Wageningen University, The Netherlands.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Gygli
authors.affiliation: Laboratory of Biochemistry, Wageningen University, The Netherlands.
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D004926:Escherichia coli; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase
keywords: flavoprotein; loop; oligomerization; phylogeny; protein engineering