Computational Investigation of Selectivity in Biosynthetic C–H Abstraction by Novel Enzymes

This dissertation describes the elucidation of the reaction mechanisms and of the sources of regio- and stereoselectivity within several novel enzymes in biosynthetic pathways with computational methods. The computational protocol utilizes both density functional theory (DFT) and molecular dynamics (MD) simulations. The details of this computational approach are described in Chapter 1. Several collaborations with experimental research groups are described in Chapters 2–5. Chapter 1 is an overview of the computational protocol utilized to study the enzyme mechanisms and selectivity, which utilizes both DFT and MD simulations. An outline of the protocol is given followed by previous examples of its usage from the Houk group. This provides the computational framework that is utilized in the remaining chapters of the dissertation. Chapter 2 describes a collaboration between our group and Prof. David Sherman’s research group, where a novel iterative P450 monooxygenase, TamI, was discovered to perform three selective oxidations (an allylic hydroxylation, epoxidation and primary hydroxylation) on a class of natural products, tirandamycins, in that specified order. Computations were performed to understand the nature of TamI’s oxidation order preference in addition to the origins of regio- and stereoselectivity of each reaction. We determined that the order of the reactions was controlled by the intrinsic free energy preference of the competing oxidations reactions while the stereoselectivity was controlled by orientation of the tirandamycin substrate within TamI, which was attributed to the hydrophobic interactions. Chapter 3 continues on the collaboration described in Chapter 2, which utilizes TamI as the basis for engineering a toolbox of biocatalysts for altered oxidative selectivity for C–H functionalization. New TamI mutants of these hydrophobic residues were discovered that demonstrated altered selectivity from the WT. Most interesting, the TamI L244A_L295V performed an additional oxidation (hydroxyl to ketone) without the aid of a co-enzyme, TamL. Computations were performed to elucidate the mechanism of this novel oxidation in TamI L244A_L295V and the molecular basis for this reactivity. In addition, the free energies of these new oxidations and selectivity were compared to the WT reaction. It was discovered that the oxidation mechanism goes through a C–H abstraction over an O–H abstraction in the rate-limiting transition state step, and the flexibility of the tirandamycin substrate within active site doe to the L244A and L295V mutations allows for the correct orientation to be accessed for this reaction to occur in the mutant and not the WT. Chapter 4 describes a collaboration between our group and Prof. David Sherman’s research group, where a novel flavin monooxygenase, PhqK, was discovered to catalyze spirocycle formation in the biosynthesis of paraherquamides. Computations were performed to explore the mechanisms and indole substituents effects, the origin of stereoselectivity, and the effects of key active site residues, R192 and D47. We determined that the mechanism was a general-acid catalyzed epoxide opening followed by a 1,2 shift with the R192 likely acting as the soured of the general acid. Chapter 5 describes a collaboration between our group and Prof. Katherine Ryan’s groups, where a novel set of pyridoxal phosphate-dependent arginine oxidases, were discovered to catalyze either desaturation or hydroxylation reactions and experiments suggested a potential role of molecular oxygen in the reactivity. We performed DFT calculations to elucidate the role of molecular oxygen in the oxidases activity and the role that water plays in the outcome of the reaction