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Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1
Jaime Fernández De Santaella1,2,3, Jin Ren1,2, Rosario Vanella1,2
1Department of Chemistry, Institute of Physical Chemistry, University of Basel, 4058 Basel, Switzerland.
Insights
We developed a new enzyme assay to screen human arginase-1 (hArg1) activity. This method identified an R21E variant with improved catalytic efficiency, useful for directed evolution and drug discovery.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human arginase-1 (hArg1) plays a crucial role in the urea cycle and is a target for therapeutic intervention.
- Efficient screening methods are needed to study hArg1 variants and identify potential drug candidates.
Purpose of the Study:
- To develop a novel, scalable enzyme cascade assay for screening human arginase-1 (hArg1) activity.
- To utilize this assay for the directed evolution and characterization of hArg1 variants.
Main Methods:
- A multi-enzyme cascade integrating hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase was designed.
- Colorimetric and fluorescent readouts were employed to monitor hArg1 activity in soluble enzymes and E. coli-displayed formats.
- A 13-member hArg1 library, focusing on mutations near catalytic residues D128 and H141, was screened.
Main Results:
- The enzyme cascade assay successfully detected and quantified hArg1 activity.
- The R21E hArg1 variant was identified, exhibiting a 13% increase in catalytic turnover rate compared to wild-type.
- The assay demonstrated scalability and suitability for high-throughput screening.
Conclusions:
- A robust and scalable one-pot, single-step assay for hArg1 activity has been established.
- This assay facilitates high-throughput screening, directed evolution of arginase libraries, and drug candidate evaluation.
- The identification of enhanced hArg1 variants opens avenues for further biochemical and therapeutic research.
Abstract:
We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of Escherichia coli. We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type. Our scalable one-pot single-step arginase assay with continuous kinetic readout is amenable to high-throughput screening and directed evolution of arginase libraries and testing drug candidates for arginase inhibition.
