Second-Coordination Sphere Effects on the Chemo-Selectivity of Nonheme Iron Dioxygenases. A Computational Study.

Abstract

Non-heme iron dioxygenases are versatile biological enzymes in all life forms, including the human body. They catalyse various chemical reactions, including the biosynthesis of natural products, e.g., hydroxyproline in mammals. However, they are also involved in the biodegradation of toxic compounds, for example, toxic cysteine in the brain. In many of these reaction pathways, a regio- or stereoselective reaction occurs, making this enzyme class of interest to the biotechnology industry for producing highvalue compounds. Many questions remain about the high selectivity patterns of non-heme iron enzymes and how the enzyme drives these mechanisms. Therefore, a detailed computational study was performed on a set of well-characterised non-heme iron dioxygenases with different functions in Nature. This dissertation focuses on three non-heme iron dioxygenases, of which a recent crystal structure was determined. These structures have been used to elucidate the selectivity patterns' reaction mechanisms and origins. In particular, the work described in this thesis shows that the second-coordination sphere of the enzyme plays a vital part in substrate binding and positioning. Moreover, electrostatic and polar interactions from the second coordination sphere often induce a local dipole moment or electric field effect and drive the reaction to an otherwise thermodynamically unfavourable pathway. My work has given insight into the intricate details of enzyme design and how regio- and chemoselectivities can be achieved. This insight can be used to engineer non-heme iron dioxygenases and give them original reactivity patterns with high selectivity for biotechnological applications. My work details computational studies on three non-heme iron dioxygenases, namely UndA (undecene biosynthesis enzyme), TmpA (2-(trimethylammonio)-ethylphosphonate dioxygenase) and AAD (aryloxyalkanoate dioxygenase). The latter enzyme is found in agricultural plants such as rice and is shown to be able to trigger the biodegradation of herbicides. Our work has given insight into the mechanism of the herbicide biodegradation and the substrate scope of the enzyme. For the UndA biosynthesis enzyme, a controversy had arisen about whether it is a mononuclear non-heme iron dioxygenase; hence models for mononuclear and dinuclear systems were created that established it as a dinuclear enzyme. Finally, the TmpA work showed that this enzyme reacts via negative catalysis, whereby a thermodynamically favourable pathway is blocked, favouring a higher energy reaction channel. My work highlights the second coordination sphere groups that induce a local electric field and guide the reaction to the wanted products.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Samuel De Visser (Supervisor) & Christopher Blanford (Supervisor)