12 research outputs found
Computational Protocol to Understand P450 Mechanisms and Design of Efficient and Selective Biocatalysts
Cytochrome P450 enzymes have gained significant interest as selective oxidants in late-stage chemical synthesis. Their broad substrate scope enables them to be good candidates for their use in non-natural reactivity. Directed evolution evolves new enzyme biocatalysts that promote alternative reactivity for chemical synthesis. While directed evolution has proven useful in developing biocatalysts for specific purposes, this process is very time and labor intensive, and therefore not easily repurposed. Computational analysis of these P450 enzymes provides great insights into the broad substrate scope, the variety of reactions catalyzed, the binding specificity and the study of novel biosynthetic reaction mechanisms. By discovering new P450s and studying their reactivities, we uncover new insights into how this reactivity can be harnessed. We discuss a standard protocol using both DFT calculations and MD simulations to study a variety of cytochrome P450 enzymes. The approach entails theozyme models to study the mechanism and transition states via DFT calculations and subsequent MD simulations to understand the conformational poses and binding mechanisms within the enzyme. We discuss a few examples done in collaboration with the Tang and Sherman/Montgomery groups toward elucidating enzyme mechanisms and rationally designing new enzyme mutants as tools for selective C–H functionalization methods
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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
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Molecular Basis for Spirocycle Formation in the Paraherquamide Biosynthetic Pathway
The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites, paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L is the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents
A shared mechanistic pathway for pyridoxal phosphate–dependent arginine oxidases
The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases
Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway
Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br /
Molecular Basis for Spirocycle Formation in the Paraherquamide Biosynthetic Pathway
The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L was the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides, and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents.
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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway
Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C─H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C─H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C─H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade
Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics
Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of
complex natural product scaffolds. These enzymes represent new tools for selectivity and
cascade reactions, facilitating direct access to core structure diversification. Recently, we
reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular
basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the
substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts,
generated by mutations at Leu101, Leu244 and/or Leu295, that alter the native selectivity, step
sequence and number of reactions catalyzed, including the engineering of a variant capable of
catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative
partner TamL. The tuned enzymes override inherent substrate reactivity enabling catalyst-
controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural
product tirandamycin. Five new, bioactive tirandamycin derivatives (6-10) were generated
through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD
simulations provide important insights on the basis of altered selectivity and underlying
biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.
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Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C–H Functionalization and Epoxidation of Tirandamycin Antibiotics
Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI