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dc.contributor.authorWrobel, Alexandra T.
dc.contributor.authorLippard, Stephen J.
dc.contributor.authorLiang, Alexandria D.
dc.date.accessioned2015-06-02T12:54:12Z
dc.date.available2015-06-02T12:54:12Z
dc.date.issued2014-05
dc.date.submitted2014-05
dc.identifier.issn0006-2960
dc.identifier.issn1520-4995
dc.identifier.urihttp://hdl.handle.net/1721.1/97142
dc.description.abstractToluene/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.en_US
dc.description.sponsorshipNational Institute of General Medical Sciences (U.S.) (Grant 2R01-GM032113)en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM008334)en_US
dc.language.isoen_US
dc.publisherAmerican Chemical Society (ACS)en_US
dc.relation.isversionofhttp://dx.doi.org/10.1021/bi500387yen_US
dc.rightsArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.en_US
dc.sourceACSen_US
dc.titleA Flexible Glutamine Regulates the Catalytic Activity of Toluene o-Xylene Monooxygenaseen_US
dc.typeArticleen_US
dc.identifier.citationLiang, Alexandria Deliz, Alexandra T. Wrobel, and Stephen J. Lippard. “A Flexible Glutamine Regulates the Catalytic Activity of Toluene o-Xylene Monooxygenase.” Biochemistry 53, no. 22 (June 10, 2014): 3585–3592. © 2014 American Chemical Societyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.mitauthorLiang, Alexandria D.en_US
dc.contributor.mitauthorWrobel, Alexandra T.en_US
dc.contributor.mitauthorLippard, Stephen J.en_US
dc.relation.journalBiochemistryen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsLiang, Alexandria Deliz; Wrobel, Alexandra T.; Lippard, Stephen J.en_US
dc.identifier.orcidhttps://orcid.org/0000-0002-2693-4982
mit.licensePUBLISHER_POLICYen_US
mit.metadata.statusComplete


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