Recent progress in intein research: from mechanism to directed evolution and applications

Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.     

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.