Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis

Polic, Vanja; Auclair, Karine

doi:10.1016/j.bmc.2014.06.034

Cited by 24 publications

(17 citation statements)

References 78 publications

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“…Co‐crystallization of 4‐F‐ l ‐tryptophan with TxtE should offer novel and detailed insights. On the other hand, substrate‐controlled regio‐selectivity has been observed in several P450 reactions . PikC hydroxylates both C12 and C10 of YC‐17 in pikromycin biosynthesis .…”

Section: Discussionmentioning

confidence: 99%

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Zuo

Zhang

Huguet‐Tapia

et al. 2016

Biotechnology Journal

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Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics.

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Section: Discussionmentioning

confidence: 99%

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Zuo

Zhang

Huguet‐Tapia

et al. 2016

Biotechnology Journal

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“…Herein, we demonstrate a method for addressing this challenge that relies on a directing group distal to the site of functionalization and overrides steric or electronic effects inherent to the substrate with the ultimate goal of achieving a P450/directing group platform that can be broadly applied without requiring extensive protein engineering for each new substrate. While such a synthetic strategy using small molecule catalysts originates from the pioneering early studies by Breslow 14,15 and has been applied in recent efforts to achieve meta -functionalizations of arenes 16,17 , the architectural complexity and unique anchoring mechanism of biological catalysts provides what is perhaps the ideal platform for engineering directed reactions to functionalize remote and unactivated structural elements 18,19,20 .…”

Section: Introductionmentioning

confidence: 99%

Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations

et al. 2015

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The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here we report the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally-guided protein and substrate engineering, and applied to the oxidation of unactivated methylene C-H bonds. Molecular dynamics and quantum mechanical calculations were employed to develop a predictive model for substrate scope, site selectivity, and stereoselectivity of PikC mediated C-H oxidation. A suite of menthol derivatives was screened computationally and evaluated through in vitro reactions where each substrate adhered to the predicted models for selectivity and conversion to product. This platform was also expanded beyond menthol-based substrates to the selective hydroxylation of a variety of substrate cores ranging from cyclic to fused bicyclic and bridged bicyclic compounds.

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“…Recently, a new strategy of substrate engineering has been explored (23,24), wherein anchoring/directing groups are chemically linked to nonsubstrate compounds. Initial studies demonstrated that the addition of these anchoring groups facilitates enzyme-substrate interactions and enables selective oxidation of C-H bonds in previously inaccessible substrates.…”

mentioning

confidence: 99%

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

Dong

Zhang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/ anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p-and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.alkylphenol | selective oxidation | chemomimetic biocatalysis | cytochrome P450 enzymes | Corynebacterium glutamicum A lkylphenols, p-, m-, and o-alkylated phenols, e.g., 1-10, (Fig. 1A) are among the most important synthetic precursors for manufacturing a vast variety of chemical products including detergents, polymers, lubricants, antioxidants, emulsifiers, pesticides, and pharmaceuticals (1). Alkylphenols are also priority environmental pollutants because they are toxic, xenoestrogenic, or carcinogenic to wildlife and humans (2, 3). The selective oxidation of the aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates for synthesizing more downstream products (4), but also in biological degradation of these environmentally hazardous compounds (5). In particular, some oxidized alkylphenols are direct structural motifs in drugs (e.g., metoprolol) (6) and bioactive ingredients of medicinal plants (Fig. 1B).Chemically, these oxidative transformations have been practiced for a long time by using superoxides, organocatalyst, high valence transition metals, or strong oxidants such as nitric acid, chromic acid, and potassium permanganate (4,7,8). However, a number of problems are persistently associated with the chemical oxidation of aliphatic C-H bonds in alkylphenols: (i) the requirement of protection/deprotection of the phenolic hydroxyl group under reaction conditions that are distinct from those for oxidation complicates the synthetic process; (ii) it is difficult to delicately control the extent of oxidations (i.e., alcohol vs. aldehyde/ketone vs. carboxylic acid); (iii) the oxidation of an aromatic C-H bond may sometimes occur when using strong oxidants; (iv) the regio-and stereoselective oxidation of an unactivated sp 3 C-H bond (in alkylphenols) remains a central challenge despite recent advances in combined use of specialized directing groups with transition met...

show abstract

Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis

Cited by 24 publications

References 78 publications

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

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