Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1

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.

Automated summary

We developed an enzyme cascade with horseradish peroxidase-based readout to screen human arginase-1 (hArg1) activity. The cascade combined four enzymes and generated colorimetric or fluorescent signals in response to hArg1 activity. By screening a curated hArg1 library, we identified a variant with enhanced catalytic turnover rate compared to wild type. Our assay is scalable and suitable for high-throughput screening and drug candidate testing for arginase inhibition.