Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation

2015 ◽  
Vol 51 (12) ◽  
pp. 2208-2224 ◽  
Author(s):  
Gheorghe-Doru Roiban ◽  
Manfred T. Reetz
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Organic Compounds ◽  
Added Value ◽  
Cytochrome P450 Enzymes ◽  
P450 Monooxygenases

Cytochrome P450 enzymes (CYPs) have been used for more than six decades as catalysts for the CH-activating oxidative hydroxylation of organic compounds with formation of added-value products.

scholarly journals MEMS directed evolution of two cytochrome P450 enzymes revealing distinct active-sites for convergent functions

2020 ◽  
Author(s):  
Li Ma ◽  
Fengwei Li ◽  
Xingwang Zhang ◽  
Hui Chen ◽  
Qian Huang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Active Sites ◽  
Enzyme Engineering ◽  
Proof Of Concept ◽  
Cytochrome P450 Enzymes ◽  
Natural Evolution ◽  
One Pot ◽  
P450 Bm3 ◽  
Protein Enzyme

AbstractDirected evolution (DE) inspired by natural evolution (NE) has been achieving tremendous successes in protein/enzyme engineering. However, the conventional ‘one-protein-for-one-task’ DE cannot match the ‘multi-proteins-for-multi-tasks’ NE in terms of screening throughput and efficiency, thus often failing to meet the fast-growing demands for biocatalysts with desired properties. In this study, we design a novel ‘multi-enzyme-for-multi-substrate’ (MEMS) DE model and establish the proof-of-concept by running a NE-mimicking and higher-throughput screening on the basis of ‘two-P450s-against-seven-substrates’ (2P×7S) in one pot. With the significantly improved throughput and hit-rate, we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes (P450 BM3 and P450cam) in laboratory. Further structural analysis of the two functionally convergent P450 variants provide important insights into how distinct active-sites can reach a common catalytic goal.

scholarly journals Genetically Tunable Enzymatic C‒H Amidation for Lactam Synthesis

2019 ◽  
Author(s):  
Inha Cho ◽  
Zhijun Jia ◽  
Frances H. Arnold
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Cytochrome P450 Enzymes ◽  
Site Selectivity

<p>Directed evolution of cytochrome P450 enzymes fine-tunes site selectivity of new-to-nature C‒H amidation for modular, sustainable and scalable preparation of enantio-enriched β-, γ- and δ-lactams.<br></p><p></p>

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

2015 ◽  
Vol 467 (1) ◽  
pp. 1-15 ◽  
Author(s):  
James B.Y.H. Behrendorff ◽  
Weiliang Huang ◽  
Elizabeth M.J. Gillam
Keyword(s):  
Cytochrome P450 ◽  
Substrate Specificity ◽  
Directed Evolution ◽  
Practical Application ◽  
Cytochrome P450 Enzymes ◽  
Level Of Activity ◽  
Modern Chemical ◽  
Stereo Selectivity ◽  
High Degree ◽  
Made In

Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

scholarly journals Site-selective enzymatic C‒H amidation for synthesis of diverse lactams

2020 ◽  
Author(s):  
Inha Cho ◽  
Zhijun Jia ◽  
Frances H. Arnold
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Chemical Engineering ◽  
Careful Examination ◽  
California Institute ◽  
Cytochrome P450 Enzymes ◽  
California Institute Of Technology ◽  
Site Selectivity ◽  
Institute Of Technology ◽  
Site Selective

<p>Please note that this work has been retracted by the authors.</p><p><br></p><p>After publication of the Report “Site-selective enzymatic C‒H amidation for synthesis of diverse lactams” in <i>Science </i>(<i>1, also linked in metadata</i>), efforts to reproduce the work showed that the enzymes do not catalyze the reactions with the activities and selectivities claimed. Careful examination of the first author’s lab notebook then revealed missing contemporaneous entries and raw data for key experiments. The authors therefore have retracted the article from <i>Science</i> and are now retracting the preprint from ChemRxiv. The original paper can be accessed by selecting “Version 1” of the preprint below, or by accessing <a href="https://doi.org/10.26434/chemrxiv.7711418.v1">https://doi.org/10.26434/chemrxiv.7711418.v1</a>. </p><p><br></p><p><b><i> Inha Cho, Zhi-Jun Jia, Frances H. Arnold*</i></b></p><p><b><i><br></i></b></p><p>Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.</p><p><br></p><p>*Corresponding author. Email: [email protected]</p><p><b><br></b></p><p><b>References</b></p><p>1. I. Cho, Z.-J. Jia, F. H. Arnold, <i>Science</i> <b>364</b>, 575 (2019). DOI: 10.1126/science.aaw9068<br></p><p><br></p><p><br></p><p>*******************************************************************************************</p><p><br></p><p>Directed evolution of cytochrome P450 enzymes fine-tunes site selectivity of new-to-nature C‒H amidation for modular, sustainable and scalable preparation of enantio-enriched β-, γ- and δ-lactams.<br></p><p></p>

scholarly journals Site-selective enzymatic C‒H amidation for synthesis of diverse lactams

2020 ◽  
Author(s):  
Inha Cho ◽  
Zhijun Jia ◽  
Frances H. Arnold
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Chemical Engineering ◽  
Careful Examination ◽  
California Institute ◽  
Cytochrome P450 Enzymes ◽  
California Institute Of Technology ◽  
Site Selectivity ◽  
Institute Of Technology ◽  
Site Selective

<p>Please note that this work has been retracted by the authors.</p><p><br></p><p>After publication of the Report “Site-selective enzymatic C‒H amidation for synthesis of diverse lactams” in <i>Science </i>(<i>1, also linked in metadata</i>), efforts to reproduce the work showed that the enzymes do not catalyze the reactions with the activities and selectivities claimed. Careful examination of the first author’s lab notebook then revealed missing contemporaneous entries and raw data for key experiments. The authors therefore have retracted the article from <i>Science</i> and are now retracting the preprint from ChemRxiv. The original paper can be accessed by selecting “Version 1” of the preprint below, or by accessing <a href="https://doi.org/10.26434/chemrxiv.7711418.v1">https://doi.org/10.26434/chemrxiv.7711418.v1</a>. </p><p><br></p><p><b><i> Inha Cho, Zhi-Jun Jia, Frances H. Arnold*</i></b></p><p><b><i><br></i></b></p><p>Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.</p><p><br></p><p>*Corresponding author. Email: [email protected]</p><p><b><br></b></p><p><b>References</b></p><p>1. I. Cho, Z.-J. Jia, F. H. Arnold, <i>Science</i> <b>364</b>, 575 (2019). DOI: 10.1126/science.aaw9068<br></p><p><br></p><p><br></p><p>*******************************************************************************************</p><p><br></p><p>Directed evolution of cytochrome P450 enzymes fine-tunes site selectivity of new-to-nature C‒H amidation for modular, sustainable and scalable preparation of enantio-enriched β-, γ- and δ-lactams.<br></p><p></p>

scholarly journals Cytochrome P450 Enzymes as Key Drivers of Alkaloid Chemical Diversification in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Trinh-Don Nguyen ◽  
Thu-Thuy T. Dang
Keyword(s):  
Cytochrome P450 ◽  
Structural Diversity ◽  
Plant Evolution ◽  
Cytochrome P450 Monooxygenases ◽  
Cytochrome P450 Enzymes ◽  
P450 Monooxygenases ◽  
Anti Cancer ◽  
Psychedelic Drugs ◽  
Cancer Agent ◽  
Alkaloid Metabolism

Plants produce more than 20,000 nitrogen-containing heterocyclic metabolites called alkaloids. These chemicals serve numerous eco-physiological functions in the plants as well as medicines and psychedelic drugs for human for thousands of years, with the anti-cancer agent vinblastine and the painkiller morphine as the best-known examples. Cytochrome P450 monooxygenases (P450s) play a key role in generating the structural variety that underlies this functional diversity of alkaloids. Most alkaloid molecules are heavily oxygenated thanks to P450 enzymes’ activities. Moreover, the formation and re-arrangement of alkaloid scaffolds such as ring formation, expansion, and breakage that contribute to their structural diversity and bioactivity are mainly catalyzed by P450s. The fast-expanding genomics and transcriptomics databases of plants have accelerated the investigation of alkaloid metabolism and many players behind the complexity and uniqueness of alkaloid biosynthetic pathways. Here we discuss recent discoveries of P450s involved in the chemical diversification of alkaloids and how these inform our approaches in understanding plant evolution and producing plant-derived drugs.

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