A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts
Abstract
Abstract Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450 BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.
- Authors:
-
[1];
[2];
[4];
[5]
- University of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, San Diego, CA (United States). Scripps Institute of Oceanography
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Department of Energy Joint Genome Institute (DOE JGI)
- Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
- Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Department of Energy Joint Genome Institute (DOE JGI)
- University of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Danmarks Tekniske University, Lyngby (Denmark); Shenzhen Institute of Advanced Technology, Shenzen (China)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Science Foundation (NSF)
- OSTI Identifier:
- 1545150
- Alternate Identifier(s):
- OSTI ID: 1526088
- Grant/Contract Number:
- AC02-05CH11231; 1RC1GM090980; F32GM125179; F32GM129960; 1341894; 1442724
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Angewandte Chemie (International Edition)
- Additional Journal Information:
- Journal Name: Angewandte Chemie (International Edition); Journal Volume: 58; Journal Issue: 30; Journal ID: ISSN 1433-7851
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; high-throughput screening; mass spectrometry; enzyme assays; biocatalysis; cytochrome P450
Citation Formats
de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., and Keasling, Jay D. A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts. United States: N. p., 2019.
Web. doi:10.1002/anie.201901782.
de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., & Keasling, Jay D. A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts. United States. https://doi.org/10.1002/anie.201901782
de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., and Keasling, Jay D. Wed .
"A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts". United States. https://doi.org/10.1002/anie.201901782. https://www.osti.gov/servlets/purl/1545150.
@article{osti_1545150,
title = {A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts},
author = {de Rond, Tristan and Gao, Jian and Zargar, Amin and de Raad, Markus and Cunha, Jack and Northen, Trent R. and Keasling, Jay D.},
abstractNote = {Abstract Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450 BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.},
doi = {10.1002/anie.201901782},
journal = {Angewandte Chemie (International Edition)},
number = 30,
volume = 58,
place = {United States},
year = {Wed May 29 00:00:00 EDT 2019},
month = {Wed May 29 00:00:00 EDT 2019}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 20 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Colloid-based multiplexed screening for plant biomass-degrading glycoside hydrolase activities in microbial communities
journal, January 2011
- Reindl, W.; Deng, K.; Gladden, J. M.
- Energy & Environmental Science, Vol. 4, Issue 8
Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme: Encoding substrates with mass tags using Nimzyme
journal, February 2012
- Deng, Kai; George, Kevin W.; Reindl, Wolfgang
- Rapid Communications in Mass Spectrometry, Vol. 26, Issue 6
Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties
journal, November 2007
- Fasan, Rudi; Chen, Mike M.; Crook, Nathan C.
- Angewandte Chemie International Edition, Vol. 46, Issue 44, p. 8414-8418
Cytochrome P450 monooxygenases: an update on perspectives for synthetic application
journal, January 2012
- Urlacher, Vlada B.; Girhard, Marco
- Trends in Biotechnology, Vol. 30, Issue 1
Identification of selectivity-determining residues in cytochrome P450 monooxygenases: A systematic analysis of the substrate recognition site 5
journal, March 2009
- Seifert, Alexander; Pleiss, Jürgen
- Proteins: Structure, Function, and Bioinformatics, Vol. 74, Issue 4
Diversity of P450 enzymes in the biosynthesis of natural products
journal, January 2012
- Podust, Larissa M.; Sherman, David H.
- Natural Product Reports, Vol. 29, Issue 10
Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)
journal, May 2014
- Deng, Kai; Takasuka, Taichi E.; Heins, Richard
- ACS Chemical Biology, Vol. 9, Issue 7
Phylogenomically Guided Identification of Industrially Relevant GH1 β-Glucosidases through DNA Synthesis and Nanostructure-Initiator Mass Spectrometry
journal, July 2014
- Heins, Richard A.; Cheng, Xiaoliang; Nath, Sangeeta
- ACS Chemical Biology, Vol. 9, Issue 9
A nanostructure-initiator mass spectrometry-based enzyme activity assay
journal, March 2008
- Northen, T. R.; Lee, J.-C.; Hoang, L.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 10, p. 3678-3683
Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS
journal, October 2015
- Deng, Kai; Guenther, Joel M.; Gao, Jian
- Frontiers in Bioengineering and Biotechnology, Vol. 3
Directed Evolution of the Fatty-Acid Hydroxylase P450 BM-3 into an Indole-Hydroxylating Catalyst
journal, April 2000
- Li, Qing-Shan; Schwaneberg, Ulrich; Fischer, Peter
- Chemistry – A European Journal, Vol. 6, Issue 9
Nootkatone—a biotechnological challenge
journal, March 2009
- Fraatz, Marco A.; Berger, Ralf G.; Zorn, Holger
- Applied Microbiology and Biotechnology, Vol. 83, Issue 1
Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3
journal, January 2005
- Sowden, Rebecca J.; Yasmin, Samina; Rees, Nicholas H.
- Organic & Biomolecular Chemistry, Vol. 3, Issue 1
A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes
journal, July 2002
- Rostovtsev, Vsevolod V.; Green, Luke G.; Fokin, Valery V.
- Angewandte Chemie International Edition, Vol. 41, Issue 14, p. 2596-2599
OpenMSI Arrayed Analysis Toolkit: Analyzing Spatially Defined Samples Using Mass Spectrometry Imaging
journal, May 2017
- de Raad, Markus; de Rond, Tristan; Rübel, Oliver
- Analytical Chemistry, Vol. 89, Issue 11
Characterization of Orphan Monooxygenases by Rapid Substrate Screening Using FT-ICR Mass Spectrometry
journal, June 2008
- Furuya, Toshiki; Nishi, Tatsunari; Shibata, Daisuke
- Chemistry & Biology, Vol. 15, Issue 6
Discovery of Potent Parthenolide-Based Antileukemic Agents Enabled by Late-Stage P450-Mediated C—H Functionalization
journal, October 2013
- Kolev, Joshua N.; O’Dwyer, Kristen M.; Jordan, Craig T.
- ACS Chemical Biology, Vol. 9, Issue 1
Acoustic deposition with NIMS as a high-throughput enzyme activity assay
journal, March 2012
- Greving, Matthew; Cheng, Xiaoliang; Reindl, Wolfgang
- Analytical and Bioanalytical Chemistry, Vol. 403, Issue 3
Controlled Oxidation of Remote sp 3 C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity
journal, November 2012
- Zhang, Kaidong; Shafer, Brian M.; Demars, Matthew D.
- Journal of the American Chemical Society, Vol. 134, Issue 45
Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation
journal, August 2001
- Farinas, Edgardo T.; Schwaneberg, Ulrich; Glieder, Anton
- Advanced Synthesis & Catalysis, Vol. 343, Issue 6-7
Directed evolution drives the next generation of biocatalysts
journal, July 2009
- Turner, Nicholas J.
- Nature Chemical Biology, Vol. 5, Issue 8
Achieving Regio- and Enantioselectivity of P450-Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure-Guided Directed Evolution
journal, June 2012
- Agudo, Rubén; Roiban, Gheorghe-Doru; Reetz, Manfred T.
- ChemBioChem, Vol. 13, Issue 10
Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio-, Stereo- and Chemoselectivity
journal, March 2009
- Seifert, Alexander; Vomund, Sandra; Grohmann, Katrin
- ChemBioChem, Vol. 10, Issue 5
Methods for the directed evolution of proteins
journal, June 2015
- Packer, Michael S.; Liu, David R.
- Nature Reviews Genetics, Vol. 16, Issue 7
Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology
journal, April 2016
- Girvan, Hazel M.; Munro, Andrew W.
- Current Opinion in Chemical Biology, Vol. 31
Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio-, Stereo- and Chemoselectivity
journal, June 2009
- Seifert, Alexander; Vomund, Sandra; Grohmann, Katrin
- ChemBioChem, Vol. 10, Issue 9
High-Throughput Method for Determining the Enantioselectivity of Enzyme-Catalyzed Hydroxylations Based on Mass Spectrometry
journal, June 2010
- Chen, Yongzheng; Tang, Weng Lin; Mou, Jie
- Angewandte Chemie, Vol. 122, Issue 31
Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates
journal, April 2018
- Sun, Huihua; Zhang, Hongfang; Ang, Ee Lui
- Bioorganic & Medicinal Chemistry, Vol. 26, Issue 7
High throughput screening of enzyme activity with mass spectrometry imaging
journal, February 2015
- de Rond, Tristan; Danielewicz, Megan; Northen, Trent
- Current Opinion in Biotechnology, Vol. 31
Impact of high-throughput screening in biomedical research
journal, March 2011
- Macarron, Ricardo; Banks, Martyn N.; Bojanic, Dejan
- Nature Reviews Drug Discovery, Vol. 10, Issue 3
Inverting the enantioselectivity of P450pyr monooxygenase by directed evolution
journal, January 2010
- Tang, Weng Lin; Li, Zhi; Zhao, Huimin
- Chemical Communications, Vol. 46, Issue 30
Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry
journal, June 2018
- Helal, Kazi Y.; Alamgir, Azmain; Berns, Eric J.
- Journal of the American Chemical Society, Vol. 140, Issue 26
Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties
journal, November 2007
- Fasan, Rudi; Chen, Mike M.; Crook, Nathan C.
- Angewandte Chemie, Vol. 119, Issue 44
A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
journal, April 1999
- Zhang, J. -H.
- Journal of Biomolecular Screening, Vol. 4, Issue 2
High-throughput strategies for the discovery and engineering of enzymes for biocatalysis
journal, October 2016
- Jacques, Philippe; Béchet, Max; Bigan, Muriel
- Bioprocess and Biosystems Engineering, Vol. 40, Issue 2
Luminogenic cytochrome P450 assays
journal, July 2006
- Cali, James J.; Ma, Dongping; Sobol, Mary
- Expert Opinion on Drug Metabolism & Toxicology, Vol. 2, Issue 4
P450 Fingerprinting Method for Rapid Discovery of Terpene Hydroxylating P450 Catalysts with Diversified Regioselectivity
journal, March 2011
- Zhang, Kaidong; El Damaty, Shady; Fasan, Rudi
- Journal of the American Chemical Society, Vol. 133, Issue 10
P450 BM3 (CYP102A1): connecting the dots
journal, January 2012
- Whitehouse, Christopher J. C.; Bell, Stephen G.; Wong, Luet-Lok
- Chem. Soc. Rev., Vol. 41, Issue 3
High-Throughput Metabolic Engineering: Advances in Small-Molecule Screening and Selection
journal, June 2010
- Dietrich, Jeffrey A.; McKee, Adrienne E.; Keasling, Jay D.
- Annual Review of Biochemistry, Vol. 79, Issue 1
Versatile synthesis of probes for high-throughput enzyme activity screening
journal, April 2013
- de Rond, Tristan; Peralta-Yahya, Pamela; Cheng, Xiaoliang
- Analytical and Bioanalytical Chemistry, Vol. 405, Issue 14
Clathrate nanostructures for mass spectrometry
journal, October 2007
- Northen, Trent R.; Yanes, Oscar; Northen, Michael T.
- Nature, Vol. 449, Issue 7165
Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3
journal, November 2003
- Peters, Matthew W.; Meinhold, Peter; Glieder, Anton
- Journal of the American Chemical Society, Vol. 125, Issue 44, p. 13442-13450
Tuning P450 Enzymes as Oxidation Catalysts
journal, March 2012
- Fasan, Rudi
- ACS Catalysis, Vol. 2, Issue 4
High-Throughput Method for Determining the Enantioselectivity of Enzyme-Catalyzed Hydroxylations Based on Mass Spectrometry
journal, June 2010
- Chen, Yongzheng; Tang, Weng Lin; Mou, Jie
- Angewandte Chemie International Edition, Vol. 49, Issue 31
Regioselective hydroxylation of steroid hormones by human cytochromes P450
journal, February 2015
- Niwa, Toshiro; Murayama, Norie; Imagawa, Yurie
- Drug Metabolism Reviews, Vol. 47, Issue 2
A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes
journal, July 2002
- Rostovtsev, Vsevolod V.; Green, Luke G.; Fokin, Valery V.
- Angewandte Chemie, Vol. 114, Issue 14, p. 2708-2711