12 research outputs found

    Computational Protocol to Understand P450 Mechanisms and Design of Efficient and Selective Biocatalysts

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    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

    A shared mechanistic pathway for pyridoxal phosphate–dependent arginine oxidases

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    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

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    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

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    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. </div

    Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

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    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

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    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. </div

    Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C–H Functionalization and Epoxidation of Tirandamycin Antibiotics

    No full text
    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
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