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A Flexible Glutamine Regulates the Catalytic Activity of Toluene o‑Xylene Monooxygenase
journal contribution
posted on 2015-12-17, 02:32 authored by Alexandria
Deliz Liang, Alexandra T. Wrobel, Stephen J. LippardToluene/o-xylene
monooxygenase (ToMO) is a bacterial
multicomponent monooxygenase capable of oxidizing aromatic substrates.
The carboxylate-rich diiron active site is located in the hydroxylase
component of ToMO (ToMOH), buried 12 Å from the surface of the
protein. A small, hydrophilic pore is the shortest pathway between
the diiron active site and the protein exterior. In this study of
ToMOH from Pseudomonas sp. OX1, the
functions of two residues lining this pore, N202 and Q228, were investigated
using site-directed mutagenesis. Steady-state characterization of
WT and the three mutant enzymes demonstrates that residues N202 and
Q228 are critical for turnover. Kinetic isotope effects and pH profiles
reveal that these residues govern the kinetics of water egress and
prevent quenching of activated oxygen intermediates formed at the
diiron active site. We propose that this activity arises from movement
of these residues, opening and closing the pore during catalysis,
as seen in previous X-ray crystallographic studies. In addition, N202
and Q228 are important for the interactions of the reductase and regulatory
components to ToMOH, suggesting that they bind competitively to the
hydroxylase. The role of the pore in the hydroxylase components of
other bacterial multicomponent monooxygenases within the superfamily
is discussed in light of these conclusions.