The structure of phenol hydroxylase and its relation to other flavoenzymes

The NADPH-dependent flavin monooxygenase phenol hydroxylase catalyses the first step in the microbial degradation pathway of phenol and several phenol derivatives. It catalyses the introduction of a hydroxyl group in the ortho-position on the aromatic ring. The crystal structure of recombinant Trichosporon cutaneum phenol hydroxylase was determined at 2.4 A resolution in complex with its cofactor, FAD, and phenol using the MIR method. The molecular model presented in this thesis contains two dimers of phenol hydroxylase and in total 1189 water molecules. It has been refined to a final R factor of 21.7% and a free R factor of 27.8%, with good stereochemistry.

The first two domains are involved in cofactor and substrate binding, while the last 200 residues fold in a separate domain of unknown function. The ADP moiety of FAD is bound by the classical ßaß motif, common to several classes of flavoenzymes. The size of the substrates is determined by the size of the substrate binding cavity. The subunits in the dimer show substantial conformational differences, which are discussed and explained in a functional perspective.

The structure of phenol hydroxylase is consistent with the earlier proposed mechanism, including hydroxyl transfer via several oxygenated flavin intermediates. The structure has given a clear answer to how substrate discrimination is achieved, in particular with respect to the difference in specificity as compared to p hydroxybenzoate hydroxylase. The structure has also given an explanation for the stabilisation of the 4a-peroxoflavin intermediate, which is achieved by exclusion of solvent during part of the catalytic reaction. In addition, possible routes for proton transfer in and out of the active site have been identified.

The existence of a large mobile segment has given some explanation as to how NADPH can bind and interact with the FAD, as well as a possible route for the substrate into and for the product to leave the active site.