SummaryMicrobial natural products represent a major source of bioactive compounds for drug discovery. Among these molecules, nonribosomal peptides (NRPs) represent a diverse class that include antibiotics, immunosuppressants, anticancer agents, toxins, siderophores, pigments, and cytostatics. The discovery of novel NRPs remains a laborious process because many NRPs consist of nonstandard amino acids that are assembled by nonribosomal peptide synthetases (NRPSs). Adenylation domains (A-domains) in NRPSs are responsible for selection and activation of monomers appearing in NRPs. During the past decade, several support vector machine-based algorithms have been developed for predicting the specificity of the monomers present in NRPs. These algorithms utilize physiochemical features of the amino acids present in the A-domains of NRPSs. In this article, we benchmarked the performance of various machine learning algorithms and features for predicting specificities of NRPSs and we showed that the extra trees model paired with one-hot encoding features outperforms the existing approaches. Moreover, we show that unsupervised clustering of 453 560 A-domains reveals many clusters that correspond to potentially novel amino acids. While it is challenging to predict the chemical structure of these amino acids, we developed novel techniques to predict their various properties, including polarity, hydrophobicity, charge, and presence of aromatic rings, carboxyl, and hydroxyl groups.

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