Virginia Tech
Biochemical characterization of Aspergillus fumigatus SidA: a flavin-dependent N-hydroxylating enzyme
Abstract
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.
Degree
thesis:*- Name thesis:degree_name
- Master of Science in Life Sciences
- Level thesis:degree_level
- masters
- Discipline thesis:degree_discipline
- Biochemistry
- Department dc:contributor.department
- Biochemistry
- Grantor dc:publisher
- Virginia Tech
- Year dc:date.issued
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Chocklett, Samuel Wyatt
- Chair dc:contributor.committeechair
-
- Sobrado, Pablo
- Committee members dc:contributor.committeemember
-
- Dean, Dennis R.
- Dolan, Erin L.
Subjects
dc:subject × 5Rights
dc:rights- Statement dc:rights
-
- In Copyright
- Licence dc:rights.uri
- Language dc:language.iso
- en_US
Identifiers
dc:identifier.*- Dc Identifier Other
- etd-12162009-143628
- OAI identifier oai:identifier
- oai:vtechworks.lib.vt.edu:10919/76915