Studies of Enzymatic and Biomimetic Halogenation and Hydroxylation in Nonheme Iron Systems

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Abstract

Halogenases are enzymes with the ability to regioselectively and stereoselectively form carbon-halogen bonds, transferring a halogen onto various carbon scaffolds forming organohalogens. These organohalogens have many biological properties, for example, antibacterial, antifungal, anti-inflammatory, anti-proliferative, anti-fouling, anti-feedant, cytotoxic, ichthyotoxic and insecticidal activity. Additionally, the halogen is highly important for biological activity and consequently pharmaceutical and agrochemical industries are interested in environmentally sustainable and economically viable methods to selectively halogenate various organic scaffolds used during organic synthesis. One such method is to use nonheme iron halogenases, which are structurally and biochemically similar to nonheme iron hydroxylases. Common to both groups is the reactive intermediate, the iron(IV)-oxo, which abstracts a hydrogen atom from a substrate. Post hydrogen atom abstraction the catalytic cycle bifurcates, producing either hydroxylated or halogenated products. Of current debate are the factors separating halogenation and hydroxylation and in this thesis we have investigated the mechanisms of the nonheme iron halogenase (HctB) and hydroxylase (P4H) using a combination of density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) to gain further insight into the bifurcation factors. The QM/MM and DFT studies on the hectochlorin biosynthesis enzyme HctB revealed that substrate binding and positioning are key for optimal substrate halogenation. Additionally, key residues (Glu223) were found to influence the charge density on the chloride ligand pushing the mechanism toward halogenation. Furthermore, the influence of substrate binding and positioning was explored further in a QM/MM and MD study on the nonheme iron hydroxylase, P4H, which hydroxylates proline residues to produce 4-hydroxyproline. The QM/MM and MD study identified that mutations to either Trp243 or Tyr140 disrupted both long and short-range interactions resulting in alterations to the enzymes regioselectivity and stereoselectivity. This study also revealed that Arg161 and Glu127 formed key interactions with the substrate, which became the focus of the next study on P4H. Together these two studies on P4H, highlighted the importance of substrate positioning and selective hydrogen bonding between the protein and substrate for correct product outcome. Additionally, we were able to explore several mutations to Trp243, Tyr140, Arg161 and Glu127, identifying mutations which resulted in changes to the enzyme̢۪s regioselectivity and stereoselectivity. Finally, in this thesis we also investigated the ability of a nonheme iron halogenase to transfer groups other than a halogen, such as nitrate and azide, using the biomimetic system , [FeIV(O)(TPA)X]+, TPA = tris(2-pyridylmethy1)amine whereby X = Cl, NO2, N3. The reaction of TPA with ethyl benzene revealed that the product distributions vary with the nature of the equatorial ligand at the metal centre. The results of this study also predict the effect of other substituents potentially opening up the application of halogenases to transferring groups other than halogens. Altogether, the studies in this thesis have looked at the different factors influencing substrate halogenation from various perspectives and have revealed the fascinating biochemistry of these enzyme̢۪s and models to perform regioselective and stereoselective reactions with potential future industrial application.

Keyword(s)

Computational; Halogenase; Hydroxylase; Nonheme; QM; QM/MM

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