STRUCTURAL ANALYSIS OF IRON (II) AND 2-(OXO)-GLUTARATE-DEPENDENT OXYGENASES WITH ALTERNATIVE REACTIVITIES

Open Access
Author:
Altmyer, Madison Jo
Area of Honors:
Science
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Amie Kathleen Boal, Thesis Supervisor
  • Ronald Albert Markle, Honors Advisor
  • Joseph M Bollinger Jr., Faculty Reader
Keywords:
  • metalloenzyme
  • oxygenase
  • hydroxylation
  • desaturases
  • isonitriles
  • crystallography
Abstract:
Iron(II) and 2-(oxo)-glutarate-dependent oxygenases constitute a major enzymatic route to oxidative functionalization of biomolecules. Enzymes in this family normally catalyze hydroxylation reactions but a small subset facilitate alternative outcomes including desaturation, halogenation, decarboxylation, and ring-closing reactions. Characterizing Fe/2OG enzymes with alternative reaction outcomes and testing novel structural mimics of a key intermediate states aids in understanding structural differences between alternative and canonical Fe/2OG systems. This thesis will compare several enzymes in this subset, including carbapenem synthase (CarC) and desaturases (e.g. P.IsnB and AmbI3) involved in the biosynthesis of isonitrile containing natural products, to the archetypal Fe/2OG hydroxylase taurine dioxygenase (TauD). As progress toward determining the structural basis for their unusual reactivity varies among these proteins, this thesis describes different stages in the characterization of these enzymes, from purification to crystallization and structural solution. Preliminary purification and crystallization protocols were explored for the desaturases AmbI3 and PIsnB, while mimics of a key mechanistic intermediate were crystallized for both TauD and CarC. This document outlines the progress in purification and crystallization of these metalloenzymes toward the ultimate goal of solving their respective x-ray structures. Finalized structures of a TauD ferryl-oxo mimic obtained during this works are described in detail.