Knowledge about the substrate scope for a given enzyme is informative for elucidating biochemical pathways and also for expanding applications of the enzyme. However, no general methods are available to accurately predict the substrate specificity of an enzyme. Pyrrolysyl-tRNA synthetase (PylRS) is a powerful tool for incorporating various noncanonical amino acids (NCAAs) into proteins, which enabled us to probe, image, rationally engineer, and evolve protein structure and function. However, the incorporation of a new NCAA typically requires the selection of large libraries of PylRS with randomized mutations at active sites, and this process requires multiple rounds of selection for each new substrate. Therefore, a single aminoacyl-tRNA synthetase with broad substrate promiscuity is ideal to facilitate widespread applications of the genetic NCAA incorporation technique. Herein, machine learning models were developed to predict the substrate specificity of PylRS to accept novel NCAAs that could be incorporated into proteins by three PylRS mutants. The models were built from a training set of 285 unique enzyme-substrate pairs of three PylRS mutants including IFRS, BtaRS, and MFRS against 95 NCAAs. The best BaggingTree (BT) model was then used for virtually screening a NCAAs library containing 1474 phenylalanine, tyrosine, tryptophan, and alanine analogues, and 156 NCAAs were predicted to be accepted by at least one of the three PylRS mutants. Then, 27 NCAAs including 24 positive and 3 negative substrates were experimentally tested for their activities, and 20 of the 24 positive substrates showed weak or strong activity and were accepted by at least one PylRS mutant, among which 11 NCAAs were never reported to be incorporated into proteins before. Three negative substrates did not show any activity. Experimental results suggested that the BT model provides a three-class classification accuracy of 0.69 and a binary classification accuracy of 0.86. This study expanded the substrate scope of three PylRS variants and provided a framework for developing machine learning models to predict substrate specificity of other PylRS variants.
Keywords: enzyme engineering; machine learning; noncanonical amino acids; pyrrolysyl-tRNA synthetase; substrate specificity.