Study of oxygenases structure and function relation

Thesis

This thesis concerns experimental studies on two structurally related Fe(II)-utilizing oxidizing enzymes – isopenicillin N synthase (IPNS) which catalyzes the key ring forming steps in the microbial biosynthesis of the penicillin antibiotics and y-butyrobetaine hydroxylase (BBOX) which catalyzes the final step in the biosynthesis of L-carnitine, a molecule vital in lipid metabolism. Chapter 1 gives an introduction to Fe(II)-utilizing oxidizing enzymes, then summarizes relevant previous studies on IPNS and BBOX. Chapter 2 describes methods for the optimized production of IPNS including in labelled form. Chapter 3 describes studies on IPNS mutants designed to disrupt the substrate binding mode and/or conformational changes during catalysis. A striking outcome of the work was the discovery of a new mode of action for IPNS, giving a product isomeric with isopenicillin N. The use of NMR spectroscopy was a unifying theme in terms of analytical studies carried out, that is protein observed NMR as described in Chapter 2 and small-molecule NMR as described in Chapter 3, where NMR studies were essential for assignment of unanticipated product structures. Further, as described in Chapter 4, NMR was used to inform on inhibitor binding to BBOX and mechanism of action. Chapter 5 provides a summary of the work and suggests avenues for future research.

Authors: Jabbary, M

• DOI: 10.5287/ora-8rz2x91y9
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English