Return to search

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

Ferrichrome 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. / Master of Science in Life Sciences

Identiferoai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/76915
Date06 January 2010
CreatorsChocklett, Samuel Wyatt
ContributorsBiochemistry, Sobrado, Pablo, Dean, Dennis R., Dolan, Erin L.
PublisherVirginia Tech
Source SetsVirginia Tech Theses and Dissertation
Languageen_US
Detected LanguageEnglish
TypeThesis, Text
Formatapplication/pdf
RightsIn Copyright, http://rightsstatements.org/vocab/InC/1.0/

Page generated in 0.0018 seconds