Title page for ETD etd-12162009-143628


Type of Document Master's Thesis
Author Chocklett, Samuel Wyatt
Author's Email Address samuelwc@vt.edu
URN etd-12162009-143628
Title Biochemical characterization of Aspergillus fumigatus SidA: a flavin-dependent N-hydroxylating enzyme
Degree Master of Science In the Life Sciences
Department Biochemistry
Advisory Committee
Advisor Name Title
Sobrado, Pablo Committee Chair
Dean, Dennis R. Committee Member
Dolan, Erin L. Committee Member
Keywords
  • Af SidA
  • hydroxylation
  • Aspergillus fumigatus
  • flavin
  • siderophore
Date of Defense 2009-12-09
Availability unrestricted
Abstract
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.
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