Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

dc.contributor.authorMachovina, Melodie M.
dc.contributor.authorUsselman, Robert J.
dc.contributor.authorDuBois, Jennifer L.
dc.date.accessioned2017-02-09T22:46:02Z
dc.date.available2017-02-09T22:46:02Z
dc.date.issued2016-08
dc.description.abstractMembers of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means.en_US
dc.description.sponsorshipNIGMS NIH HHS (R01 GM090260)en_US
dc.identifier.citationMachovina, Melodie M, Robert J Usselman, and Jennifer L DuBois. "Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations." Journal of Biological Chemistry 291, no. 34 (August 2016): 17816-17828. DOI:https://dx.doi.org/10.1074/jbc.M116.730051.en_US
dc.identifier.issn1083-351X
dc.identifier.urihttps://scholarworks.montana.edu/handle/1/12586
dc.language.isoen_USen_US
dc.rights"This research was originally published in Journal of Biological Chemistry. Machovina, Melodie M, Robert J Usselman, and Jennifer L DuBois. "Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations." Journal of Biological Chemistry 291, no. 34 (August 2016) © the American Society for Biochemistry and Molecular Biology.""en_US
dc.titleMonooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenationsen_US
dc.typeArticleen_US
mus.citation.extentfirstpage17816en_US
mus.citation.extentlastpage17828en_US
mus.citation.issue34en_US
mus.citation.journaltitleJournal of Biological Chemistryen_US
mus.citation.volume291en_US
mus.data.thumbpage6en_US
mus.identifier.categoryChemical & Material Sciencesen_US
mus.identifier.doihttps://dx.doi.org/10.1074/jbc.M116.730051en_US
mus.relation.collegeCollege of Letters & Scienceen_US
mus.relation.departmentChemistry & Biochemistry.en_US
mus.relation.universityMontana State University - Bozemanen_US

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