The Functional and Structural Characterization of Novel Diiron Oxidases

Open Access
- Author:
- Fitzpatrick, Lara
- Area of Honors:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Joseph M Bollinger, Jr., Thesis Supervisor
David D Boehr, Thesis Honors Advisor
Amie Kathleen Boal, Faculty Reader - Keywords:
- VarO
Desaturase
Diiron Enzyme
Heme Oxygenase-like
Crystallography
C-H Activation
Spectroscopy - Abstract:
- The heme oxygenase-like diiron oxygenases/oxidases (HDOs) are an emerging superfamily of O2-activating metalloenzymes known for their ability to catalyze difficult oxidative chemistry within different organic metabolites on pathways to antibiotics, biofuels, and anticancer compounds. Structurally characterized members of this family like the N-oxygenase SznF, the nitrile forming AetD, and the desaturase-lyase BesC, exhibit a solvent-accessible active site and flexible loops within their core structure that contribute to their intrinsic cofactor instability, a trait that sets them apart from the well-characterized ferritin-like diiron oxygenases/oxidases (FDOs). Recent results on a newly recognized member of this family, VarO, from S. varsoviensis (svVarO), demonstrate its desaturase reactivity in inserting a C-C double bond between C3 and C4 within the L-arginine residue that exists in APR/GPR tripeptides core of the potentially anticancer compounds thiovarsolins A and C. To do so, svVarO employs a μ-(hydro)peroxo-Fe2(III/III) complex as the initiating intermediate of the reaction. Downstream intermediates responsible for the required hydrogen atom abstraction (HAA) steps have not been captured. Moreover, key mechanistic details of its reaction mechanism remain elusive due to the absence of its three-dimensional structure. In this regard, the identification of a new homologue, S. cellulosum (scVarO), that shares a 65% sequence identity to svVarO within a similar biosynthetic gene cluster opens up the possibility to better understand desaturation by diiron enzymes. It has been hypothesized that this homologue acts as a desaturase in the biosynthesis of thiovarsolins and shares the same structural details as the characterized members of the HDOs. Using Liquid Chromatography Mass Spectrometry (LC-MS) the formation of the desaturated product from the GPR tripeptide core was confirmed, which speaks to the similarity between svVarO and scVarO. Attempts to detect intermediates in the desaturation reaction of scVarO by UV-visible stopped-flow absorption spectroscopy were not immediately successful, but ongoing efforts are directed towards this goal. The scVarO enzyme was successfully crystallized, and x-ray diffraction data was obtained, allowing for the solution of its three-dimensional structure in the apo form to a resolution of 3.0 Å. The structure reveals a core similar to that seen in the better characterized HDOs. The svVarO protein was then crystallized, and x-ray diffraction data was obtained to a resolution of 2.6 Å after cocrystallization with APR, resulting in a structure having the substrate present in the active site. These results not only advance our understanding of this fundamental reactivity in biology but also move us forward to the use of HDOs for biocatalysis applications.