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

Narayan, Alison R. H.; Jiménez‐Osés, Gonzalo; Liu, Peng; Negretti, Solymar; Zhao, Wanxiang; Gilbert, Michael M.; Ramabhadran, Raghunath O.; Yang, Yong; Furan, Lawrence R.; Li, Zhe; Podust, L.M.; Montgomery, John; Houk, K. N.; Sherman, David H.

doi:10.1038/nchem.2285

Cited by 102 publications

(114 citation statements)

References 59 publications

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“…Thus, although this computationally inexpensive docking method can provide insight into FDH selectivity, more computationally rigorous methods such as molecular dynamics simulations (120) could improve predictive abilities for FDH substrate specificity and selectivity.…”

Section: Fdh Substrate Activity Profilingmentioning

confidence: 99%

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Andorfer

Lewis

2018

Annu. Rev. Biochem.

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Flavin dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes has been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

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Section: Fdh Substrate Activity Profilingmentioning

confidence: 99%

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Andorfer

Lewis

2018

Annu. Rev. Biochem.

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“…20,22 Consequently, much of our recent work has focused on the discovery and biochemical/structural characterization of new P450s involved in the biosynthesis of bacterial natural products. 31–35 As we have demonstrated with PikC, 36–38 these fundamental studies serve as a key starting point to guide future efforts to modulate the substrate scope and selectivity properties of P450 enzymes. Engineered biosynthetic P450s may ultimately be employed in vitro or in vivo as biocatalysts for the efficient production of novel biologically active compounds.…”

mentioning

confidence: 96%

“…36 More recently, we have shown that PikC can accept a variety of other substrates attached to structurally diverse non-sugar anchors containing the N , N -dimethylamino moiety. 37,38 Although such an explicit anchoring mechanism has not been clearly established for other P450s that act on macrolide substrates, the presence of one or more sugar moieties is typically required for substrate recognition, leading in turn to effective binding and catalysis. Conversely, other P450s involved in macrolide biosynthetic pathways (e.g., EryF) strictly act on aglycone intermediates prior to glycosylation.…”

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confidence: 99%

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

et al. 2016

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Cytochrome P450 monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and x-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

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“…in regio-or stereoselective manners (27)(28)(29). However, these approaches are not completely biological because the generation, installation, and offloading of anchoring groups still rely on chemical synthesis.…”

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

Cited by 102 publications

References 59 publications

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

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

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