Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay

Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies...

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Bibliographic Details
Published in:ACS chemical biology 2017-01, Vol.12 (1), p.191-199
Main Authors: Raad, Markus de, Modavi, Cyrus, Sukovich, David J, Anderson, J. Christopher
Format: Article
Language:English
Subjects:
Arabidopsis Proteins - chemistry
Arabidopsis Proteins - genetics
Catechol O-Methyltransferase - chemistry
High-Throughput Nucleotide Sequencing - methods
Methylation
Methyltransferases - chemistry
Methyltransferases - genetics
Substrate Specificity
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Summary:Currently, the identification of new genes drastically outpaces current experimental methods for determining their enzymatic function. This disparity necessitates the development of high-throughput techniques that operate with the same scalability as modern gene synthesis and sequencing technologies. In this paper, we demonstrate the versatility of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA) and its ability to support high-throughput data acquisition through next-generation sequencing (NGS). Utilizing methyltransferases, we highlight DLEnCA’s ability to rapidly profile an enzyme’s substrate specificity, determine relative enzyme kinetics, detect biosynthetic formation of a target molecule, and its potential to benefit from the scales and standardization afforded by NGS. This improved methodology minimizes the effort in acquiring biosynthetic knowledge by tying biochemical techniques to the rapidly evolving abilities in sequencing and synthesizing DNA.
ISSN:1554-8929
1554-8937
DOI:10.1021/acschembio.6b00652