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Catalytic Site Atlas

CSA LITERATURE entry for 1foh

E.C. namephenol 2-monooxygenase
SpeciesTrichosporon cutaneum (Yeast)
E.C. Number (IntEnz) 1.14.13.7
CSA Homologues of 1foh1pn0,2dkh,2dki,
CSA Entries With UniProtID P15245
CSA Entries With EC Number 1.14.13.7
PDBe Entry 1foh
PDBSum Entry 1foh
MACiE Entry 1foh

Literature Report

IntroductionPhenol 2-monooxygenase, also known as phenol hydroxylase (PHHY) belongs to a class of flavin enzymes commonly referred to as the aromatic hydroxylases. It catalyses the conversion of simple phenols to their o-diol derivatives. The enzyme is a homodimer where each monomer of 76kDa contains noncovalently bound flavin adenine dinucleotide (FAD) The enzyme can hydroxylate simple, amino, methyl, hydroxy and halogen phenols, although it does not show any activity towards phenols carrying carboxyl groups on the benzene nucleus or its side chain.
Next to carbohydrates, phenolic compounds are the second most abundant group of natural products, many are also being produced by industry, with many of these being resistant to degradation. The reaction catalysed by phenol hydroxylase are of particular interest for industrial waste detoxification.
MechansimIt has been propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.
The catalytic mechanism of PHHY is complex, involving FAD and three substrates: molecular oxygen, phenol and NADPH. The overall mechanism is of the type bi-uni-uni-bi ping pong. While in the past the mechanism has been compared to p-hydroxy-benzoate hydroxylase, the enzymes are now though to operate differently.
The flavin cofactor is first reduced by NADPH. After releasing NADP+, the reduced flavin reacts with oxygen to form C4a-hydroperoxyflavin that it used to hydroxylate the substrate, phenol, or its derivatives at the ortho position. The immediate product undergoes further change to form C4a-hydroxyflavin. The enzyme finishes the reaction cycle by releasing a molecule of water to return the flavin to the oxidised state.
Phenol is suitably placed for attack at the ortho position through a hydrogen bond to Tyr289. This residue is also thought to control FAD conformation, and therefore direct FAD reduction. Modelling suggests that Tyr289 is also able to form a hydrogen bond to the oxygen of C4a-peroxoflavin intermediate. Asp54 together with Arg281forms a hydrogen bond to the phenolic oxygen, thereby increasing its partial negative charge, the side chain could also stabilise the positive charge on the substrate developed in the transition state. However, when these residue are mutated, their mutant forms do not decrease catalytic activity dramatically. The one residue which has been implicated in catalysis by mutagenesis, Pro364, is thought to use its carbonyl backbone to hydrogen bond to phenol substrate and so direct nucleophilic attack of the hydroperoxide at C2.
Reaction

Catalytic Sites for 1foh

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
ProA364365macie:mainChainAmideThe residue's backbone carbonyl is implicated in directing nucleophilic attack of the phenol C2 at C4a- hydroperoxyflavin.
AspA5455macie:sideChainThe residue is thought to act with Arg281 within the active site to polarise the phenolic OH bond through hydrogen bonding interactions, activating the oxygen towards conjugate attack at the C4a- hydroperoxyflavin intermediate. These residues, however, do not fulfil the role of a general base towards the phenol substrate.
ArgA281282macie:sideChainThe residue is thought to act with Asp54 within the active site to polarise the phenolic OH bond through hydrogen bonding interactions, activating the oxygen towards conjugate attack at the C4a- hydroperoxyflavin intermediate. These residues, however, do not fulfil the role of a general base towards the phenol substrate.
TyrA289290macie:sideChainThe residue affects the equilibrium between the two conformations of FAD and is thought to help the flavin move into a conformation necessary for reduction by NADPH.

Literature References

Notes:
Xu D
Studies of the mechanism of phenol hydroxylase: effect of mutation of proline 364 to serine.
Biochemistry 2002 41 13627-13636
PubMed: 12427024
Xu D
Studies of the mechanism of phenol hydroxylase: mutants Tyr289Phe, Asp54Asn, and Arg281Met.
Biochemistry 2001 40 12369-12378
PubMed: 11591156
Enroth C
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.
Structure 1998 6 605-617
PubMed: 9634698
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