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Catalytic Site Atlas

CSA LITERATURE entry for 1dii

E.C. name4-methylphenol dehydrogenase (hydroxylating)
SpeciesPseudomonas putida (Bacteria)
E.C. Number (IntEnz) 1.17.99.1
CSA Homologues of 1dii1diq,1e0y,1wve,1wvf,
CSA Entries With UniProtID P09788
CSA Entries With EC Number 1.17.99.1
PDBe Entry 1dii
PDBSum Entry 1dii
MACiE Entry M0141

Literature Report

Introduction4-Cresol dehydrogenase is a flavocytochrome c protein. It is the first enzyme in the protocatechuate metabolic pathway and is responsible for the degradation of toxic phenol p-cresol. The active site is buried deeply in the enzyme's interior. The route of substrate access has been shown to follow a swinging gate mechanism.
MechansimThe enzyme first catalyses the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilising one atom of oxygen derived from water and yielding one molecule of reduced FAD.
An enzymic base, Tyr473 removes the proton from the hydroxyl group of p-cresol and a hydride ion is transferred from the methyl group to the N5 atom of FAD. This results in a quinone methide intermediate which then undergoes nucleophilic attack by water at the methylene group, yielding p-hydroxybenzyl alcohol. Another tyrosine; Tyr95, is important in the catalytic mechanism, it is responsible for the the orientation and stabilisation of the substrate during the reaction.
His436 may also form part of a proton relay system to deprotonate transiently the catalytic tyrosine prior to proton abstraction from p-cresol to form the quinone-methide intermediate. A water molecule is positioned close to the methyl group of the substrate is suitably located for nucleophilic attack in the hydroxylation step of the mechanism, this could be activated by either Glu380 or Glu427 through polarisation or depolarisation.
A key feature of the active site is Arg474 which can use its gaunidinium group to stabilise the negative charge that develops at the N1/O2 locus in the hydroquinone and semiquinone intermediates during catalysis. A hydrogen bonding network between Asp440 and Tyr384 may assist the aspartic acid group to abstract a proton during the self-catalysed covalent flavinylation process.
Reaction

Catalytic Sites for 1dii

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
TyrA473473macie:sideChainThe residue acts as a general base towards the p-cresol group. It is activated for catalysis by His436.
GluA380380macie:sideChainThe residue activates a water molecule to act as a nucleophile at the substrate methyl group by abstracting a proton. It has been hypothesised that the residue is deprotonated through a proton relay with Tyr367 and Glu286.
HisA436436macie:sideChainThe residue is correctly aligned to abstract a proton from Tyr473, and therefore activate the phenolic oxygen to act as a general base towards the p-cresol substrate.
ArgA474474macie:sideChainThe positively charged gaunidinium group stabilises the negative charge that develops at the N1/O2 locus in the hydroquinone and semiquinone intermediates during catalysis

Literature References

Notes:
Cunane LM
Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism.
J Mol Biol 2000 295 357-374
PubMed: 10623531
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