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Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

ABSTRACT: Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

SUBMITTER: McDonald AD

PROVIDER: S-EPMC9448781 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

McDonald Allwin D AD Higgins Peyton M PM Buller Andrew R AR

Nature communications 20220906 1

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engi ...[more]

PMID: 36068220

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Project description:We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Dataset Information

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

Publications

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

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