8 research outputs found
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 Rfactor of 21.7% and a free Rfactor 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 ĂaĂ 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
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