Biochemical characterization of Aspergillus fumigatus SidA: a flavin-dependent N-hydroxylating enzyme

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dc.contributor.authorChocklett, Samuel Wyatten
dc.contributor.committeechairSobrado, Pabloen
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.committeememberDolan, Erin L.en
dc.contributor.departmentBiochemistryen
dc.date.accessioned2017-04-04T19:50:17Zen
dc.date.adate2010-01-06en
dc.date.available2017-04-04T19:50:17Zen
dc.date.issued2009-12-09en
dc.date.rdate2016-09-27en
dc.date.sdate2009-12-16en
dc.description.abstractFerrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus during infection. This siderophore includes N5-hydroxylated L-ornithine in the peptide backbone that serve as iron chelators. Af SidA is the L-ornithine N5-hydroxylase, which performs the first enzymatic step in the biosynthesis of ferrichrome. In this study, Af SidA was recombinantly expressed and purified as a soluble tetramer with a bound FAD cofactor. The enzyme demonstrated typical Michaelis-Menten kinetics in a product formation assay with respect to L-ornithine, but similar experiments as a function NADH and NADPH indicated inhibition at high coenzyme concentrations. Af SidA is highly specific for its substrate; however, it is promiscuous with respect to its coenzyme requirement. A multi-functional role of NADPH is observed since NADP+ is a competitive inhibitor with respect to NADPH and steady-state kinetic experiments indicate that Af SidA forms a ternary complex with NADP+ and L-ornithine for catalysis. Furthermore, in the absence of substrate, Af SidA forms a stable C4a-(hydro)peroxyflavin intermediate that is stable on the second time scale. Af SidA is also inhibited by several halides and the arginine-reactive reagent, phenylglyoxal. Biochemical comparison of Af SidA to other flavin-containing monooxygenases reveal that Af SidA likely proceeds by a sequential-ordered mechanism.en
dc.description.degreeMaster of Science in Life Sciencesen
dc.identifier.otheretd-12162009-143628en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12162009-143628/en
dc.identifier.urihttp://hdl.handle.net/10919/76915en
dc.language.isoen_USen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectAf SidAen
dc.subjecthydroxylationen
dc.subjectAspergillus fumigatusen
dc.subjectflavinen
dc.subjectsiderophoreen
dc.titleBiochemical characterization of Aspergillus fumigatus SidA: a flavin-dependent N-hydroxylating enzymeen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Science in Life Sciencesen

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