Computational Study of the reaction mechanism of the DNA repair enzyme AlkB

Abstract

ABSTRACTThe University of ManchesterFaculty of Engineering and Physical SciencesABSTRACT OF THESIS submitted by Luis Enrique Gonzalez-Ovalle for the degree of Master of Philosophy (MPhil) and entitled "Computational Study of the reaction mechanism of the DNA repair enzyme AlkB"Date of submission: 08/09/2011The AlkB enzyme is one of the most vital nonheme enzymes in the DNA repair mechanism in humans. Because of its importance as a promiscuous substrate recognition enzyme, it is the subject of scientific research into drug development and cancer treatment. However, currently little is known on the catalytic mechanism by the enzyme and the active oxidant in the reaction mechanism. To elucidate the active oxidant and establish a catalytic cycle we performed a series of computational studies, and, in particular, used the quantum mechanics / molecular mechanics (QM/MM) method. A novel mechanism was found, which starts from an iron(IV)-oxo species, whereby an internal rotation of the iron(IV)-oxo group initiated the reaction mechanism and brings the oxidant in close proximity with the substrate. Subsequently, a hydrogen atom abstraction by the iron(IV)-oxo species happens prior to hydroxyl rebound to form hydroxylation products. Our computational study predicts a rate determining hydrogen atom abstraction reaction step followed by concerted rebound to form products. This mechanism shows dramatic differences with other nonheme iron-oxygenases, where no internal rotation is needed and that proceed with significant rebound barriers. The studies also highlight the effects of protein on the reaction mechanism.In a second set of calculations, we investigated a novel biomimetic model of the nonheme iron enzyme cysteine dioxygenase using density functional theory methods. These systems were recently synthesised and displayed reactivity differences in the presence of chloride as compared to triflate anions. In order to understand these differences, we performed a density functional study and explain the origin of this reactivity difference. Thus, a bulky triflate group causes steric interactions with the isopropyl side groups of the equatorial ligand and consequently thiophenol cannot bind in the favourable equatorial position. As a result, triflate binding cannot give dioxygenation of thiophenol and thiophenyl radical are formed leading to PhSSPh products instead. Our calculations are in good agreement with experimental observation. Finally, recent studies of the Sorokin group revealed a nonheme diiron phthalocyanine complex capable of hydroxylating the very strong C-H bond in methane. We did a density functional theory study on the mechanism and origin of this efficient reactivity. The exploratory results give a low hydrogen abstraction barrier of 7.9 kcal mol-1 from methane by this oxidant that via an concerted mechanism leads to methanol products. The barrier obtained from this study is significantly lower than that found by an iron(IV)-oxo porphyrin cation radical species, which was 26.5 kcal mol-1. Therefore, N-bridged diiron complexes are more catalytically active than iron-porphyrins.

Date of Award	31 Dec 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Samuel De Visser (Supervisor)