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 industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate 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 phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols.


RESULTS
The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure 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 nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second 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 solvent during the hydroxylation reaction.


CONCLUSIONS
Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.
Enroth
C
C
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Neujahr
H
H
Schneider
G
G
Lindqvist
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003461:Crystallography; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D019800:Phenol / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014250:Trichosporon / Q000201:enzymology
D005420:Flavoproteins; D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D019800:Phenol; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase