THE CRYSTAL-STRUCTURE OF PHENOL HYDROXYLASE IN COMPLEX WITH FAD AND PHENOL PROVIDES EVIDENCE FOR A CONCERTED CONFORMATIONAL CHANGE IN THE ENZYME AND ITS COFACTOR DURING CATALYSIS

Background: The synthesis of phenolic compounds as by-products of indu strial reactions poses a serious threat to the environment. Understand ing the enzymatic reactions involved in the degradation and detoxifica tion of these compounds is therefore of much interest. Soil-living yea sts use flavin adenine dinucleotide (FAD)-containing enzymes to hydrox ylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy , The phenol hydroxylase from Trichosporon cutaneum hydroxylates pheno l to catechol. Phenol is the best substrate, but the enzyme also accep ts simple hydroxyl-, amino-, halogen-or methyl-substituted phenols, Re sults: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 Angstrom resolution. The struct ure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nu cleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and sec ond domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation, This conformational flexibility allows the substrate to gain access to the active site and excludes so lvent during the hydroxylation reaction. Conclusions: Two of the domai ns of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flav in-containing aromatic hydroxylases that share the same overall fold, in spite of targe differences in amino acid sequences and chain length . The structure of phenol hydroxylase is consistent with a hydroxyl tr ansfer mechanism via a peroxo-FAD intermediate. We propose that a move ment of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.