Trauger J W, Kohli R M, Mootz H D, Marahiel M A, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature. 2000 Sep 14;407(6801):215-8. doi: 10.1038/35025116.
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.
在多结构域巨型合成酶合成许多大环天然产物的过程中，羧基末端硫酯酶（TE）结构域参与环化和产物释放；然而，尚未确定TE结构域是否能够独立于上游结构域催化大环化（以及对称环肽情况下的延伸）。由于无法将TE环化步骤与早期的链组装步骤分离，因此无法确定TE底物特异性，而这对于新化合物的工程生物合成至关重要。在此，我们报告从短杆菌酪肽合成酶中切除的TE结构域能够高效催化十肽硫酯环化形成抗生素短杆菌酪肽A，并且能够催化五肽硫酯二聚化，随后环化形成抗生素短杆菌肽S。通过系统地改变十肽硫酯底物并比较环化速率，我们还表明只有两个残基（十肽两端各一个）对环化至关重要。这种特异性概况表明，短杆菌酪肽合成酶TE以及类似的许多其他TE结构域，将能够环化并释放由工程化酶组装线产生的广泛的新底物和产物。
