CRYSTAL-STRUCTURES AND INHIBITOR BINDING IN THE OCTAMERIC FLAVOENZYMEVANILLYL-ALCOHOL OXIDASE - THE SHAPE OF THE ACTIVE-SITE CAVITY CONTROLS SUBSTRATE-SPECIFICITY

Background: Lignin degradation leads to the formation of a broad spect rum of aromatic molecules that can be used by various fungal micro-org anisms as their sole source of carbon. When grown on phenolic compound s, Penicillium simplicissimum induces the strong expression of a flavi n-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkylphenols. VAO is a member of a novel class of widely distribu ted oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the dete rmination of the structure of VAO in order to shed light on the most i nteresting features of these novel oxidoreductases, such as the functi onal significance of covalent flavinylation and the mechanism of catal ysis. Results: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elonga ted cavity on the si side of the flavin molecule. Three residues, Tyr1 08, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed wi th the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bea ring aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. Conclusions: The shape of the active-site cavity contr ols substrate specificity by providing a 'size exclusion mechanism'. I nside the cavity, the substrate aromatic ring is positioned at an angl e of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by subs trate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect subs trate oxidation or reduction via hydride transfer.