Natural and Non-natural Reactivity of Heme-Thiolate Proteins

Abstract

This thesis describes the isolation and characterization of a novel and highly expressed heme-thiolate peroxygenase, MroAPO, from the fungus Marasmius rotula ATCC 76396. Sequence analyses and catalysis studies have shown MroAPO to be distinctive from its European counterpart from Marasmius rotula DSM 25031. Both MroAPO from M. rotula ATCC 76395 and DSM 25031, like AaeAPO from Agrocybe aegerita, share the ability to catalyze P450-like monooxygenation of various organic compounds, requiring only hydrogen peroxide as a co-substrate. In this dissertation ATCC 76395 MroAPO is preliminarily characterized and unique cyclopropanation and N-H insertion reactions are characterized for both ATCC 76395 MroAPO and other heme proteins including chloroperoxidase from Caldariomyces fumago and synthetic de novo heme-bound peptides.

In chapter 1, the reactivity and mechanism of heme-thiolate enzymes is reviewed, along with comparisons of APO, CPO and CYP enzymes. A summary of recombinant production of APO enzymes is also provided. In chapter 2, ATCC 76395 MroAPO is characterized and compared to DSM 25031 MroAPO. The protein from ATCC 76395 was cultivated and isolated from the fungal medium and was found to be a heme-thiolate peroxygenase similar to MroAPO from the DSM 25031 strain. Mass spectrometry revealed a number of point mutations, including a phenylalanine to tyrosine mutation adjacent to the heme and near the active site of the protein. The reactivities of ATCC 76395 MroAPO with various oxidants are compared and the mutations between ATCC 76395 MroAPO and DSM 25031 are discussed. Chapter 3 is focused on the reactivities of ATCC 76395 MroAPO, alkaline CPO, microperoxidase-11 and de novo heme-bound synthetic proteins for cyclopropanation and N-H insertion. It was found that alkaline CPO surpassed both natural and engineered heme proteins with >99% efficiency for aniline N-H insertion conversion. Chapter 4 is an examination of UV-Vis, stopped-flow and EPR spectroscopies to isolate and characterize the active intermediate involved in N-H insertion reactions with alkaline CPO. It was found that styrene cyclopropanation and N-H insertion appear to proceed through different active intermediates. Finally, chapter 5 reports various attempts to produce novel APO proteins using wild type fungi and various recombinant cellular factories.