Understanding Activity-Stability Tradeoffs in Biocatalysts by Enzyme Proximity Sequencing

Abstract

Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we developed Enzyme Proximity-Seq (EP-Seq), a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We used EP-Seq to analyze how 6,387 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase (DAOx) from R. gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. EP-Seq can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.

Decoding Stability and Epistasis in Human Myoglobin by Deep Mutational Scanning and Codon-level Machine Learning

“…We were interested in how well yeast display expression level measured by DMS could be correlated with thermodynamic stability of soluble versions of the same protein sequences. In previous work, we showed how yeast surface display levels largely correlated with computationally predicted ΔΔGfolding, supporting the idea that yeast secretion and display pathways select and filter proteins based on thermodynamic folding stability (26).…”

“…These sites contributed to substantial variability of the negative trend (Figure S7 A). These data highlight the ability of DMS to shed light onto activity-stability trade-offs (26).…”

“…We note that there is evidence that yeast surface display expression level is generally correlated with protein folding stability (23)(24)(25)(26). Thermodynamically destabilizing mutations yield lower detectable expression levels because less stably folded domains are susceptible to yeast quality control mechanisms which hinder the secretion of unstable variants.…”

Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

“…For example, human DAAOx is involved in regulating brain levels of D-Serine, a neuromodulator and agonist of the NMDA receptor implicated in schizophrenia(3) , (4). DAAOx has been further used in biosensors for measuring levels of D-Amino acids in food products (5), and in the context of engineered biological therapeutics variants of DAAOx are considered candidates for cancer therapy (6) , (7)(8)(9). In oxidative anticancer therapy, the generation of reactive oxygen species 1 (i.e.…”

“…34 DAAOx has been further used in biosensors for measuring levels of D-amino acids in food products, 5 and in the context of engineered biological therapeutics variants of DAAOx are considered candidates for cancer therapy. [6][7][8][9] In oxidative anticancer therapy, the generation of reactive oxygen species (i.e. H 2 O 2 ) upon conversion of prodrug substrates can generate therapeutic cytotoxicity based on pre-sensitization of cancer cells to oxidative stress.…”