Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase

Abstract

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single‐atom replacement in comparison to tyrosine. The arPTE‐(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12‐fold increase in kcat and 3.2‐fold enhancement in kcat/KM at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

Machine Learning-Guided Protein Engineering

“…These substitutions can be used for diverse purposes, ranging from tailoring the structural, physical, and dynamic properties of specific sites to introducing new properties by adding carefully designed UAAs. Such additions can be used to enhance enzyme activity or elucidate the enzyme reaction mechanisms . While there are some emerging ML-based tools for rational design of UAA sites, further research and development are needed to make this approach widely applicable in enzyme engineering.…”

“…Such additions can be used to enhance enzyme activity or elucidate the enzyme reaction mechanisms. 261 While there are some emerging ML-based tools for rational design of UAA sites, 262 further research and development are needed to make this approach widely applicable in enzyme engineering.…”

Rational incorporation of any unnatural amino acid into proteins by machine learning on existing experimental proofs

“…More recently, this technique has been expanded to transgenic animals including worms, fruit flies, zebrafish, and mice [3] . Since proteins are mostly composed of 20 canonical or natural amino acid (NAA) residues, the UAA substitution of a specific NAA residue, also termed as the NAA → UAA mutation, can render the protein new features or altered functions [4] , such as photo-crosslinking [5] , biorthogonal labelling [6] , site-specific conjugation [7] or improved enzymatic activities [8] . The reported UAA-incorporated proteins could serve as a good database resource for investigating the rules of UAA substitutions.…”