Directed Evolution of P 450 BM 3 into a <i>p</i>‐Xylene Hydroxylase

Dennig, Alexander; Marienhagen, Jan; Ruff, Anna Joëlle; Guddat, Lukas; Schwaneberg, Ulrich

doi:10.1002/cctc.201200092

Cited by 44 publications

(54 citation statements)

References 22 publications

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“…This observation is consistent with previous studies reporting aromatic hydroxylation of substrates like mono-substituted benzenes and o-xylene by P450 BM3 variants containing phenylalanine at position 87. 32,34 Interestingly, no benzylic hydroxylation was observed for 3.…”

Section: Application To Other Structurally Related Nsaids Mefenamic Amentioning

confidence: 90%

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Venkataraman

Verkade-Vreeker

Capoferri

et al. 2014

Bioorganic & Medicinal Chemistry

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Section: Application To Other Structurally Related Nsaids Mefenamic Amentioning

confidence: 90%

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Venkataraman

Verkade-Vreeker

Capoferri

et al. 2014

Bioorganic & Medicinal Chemistry

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“…However, this biocatalytic system was not able to oxidize o-alkylphenols. Interestingly, recent work has demonstrated that P450 BM3 can be engineered to hydroxylate alkylbenzenes to corresponding alkylphenols (42,43). Should these biocatalysts be coupled to the current alkylphenol oxidation system, an extended biochemical route from alkylbenzenes to oxidized alkylphenols could be created.…”

Section: Discussionmentioning

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...

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“…Focused mutagenesis is mainly used in semi-rational protein design experiments [14] to improve localized properties such as activity [9,[19][20][21], selectivity [22][23][24] and, less-frequently, thermal resistance [15,25,26]. The prerequisite is a crystal structure or a homology model to identify the 'beneficial' residues that are subsequently randomized [27].…”

Section: State Of the Art And Challengesmentioning

confidence: 99%

To get what we aim for – progress in diversity generation methods

2013

Self Cite

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Protein re‐engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high‐throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized ‘to get what we aim for’ through ‘optimal diversity’ generation.

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Directed Evolution of P 450 BM 3 into a p‐Xylene Hydroxylase

Cited by 44 publications

References 22 publications

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

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

To get what we aim for – progress in diversity generation methods

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