Harris Karen S, Durek Thomas, Kaas Quentin, Poth Aaron G, Gilding Edward K, Conlan Brendon F, Saska Ivana, Daly Norelle L, van der Weerden Nicole L, Craik David J, Anderson Marilyn A
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
Nat Commun. 2015 Dec 18;6:10199. doi: 10.1038/ncomms10199.
Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.
环肽是多种多样的植物骨架环化肽，作为药物支架已引起人们的关注，但其生物合成起源的基本原理仍然难以捉摸。骨架环化是环肽生物合成中关键的酶介导步骤，并赋予所得环肽一定程度的稳定性。此外，环化对于工程肽来说也是可取的。在此，我们报告了从产环肽的植物近缘老鹳草中鉴定出四种天冬酰胺基内肽酶（AEPs），这些蛋白酶与环化有关。我们重组表达了OaAEP1b，发现它通过将前体肽底物的C端切割与骨架环化偶联，优先作为环化酶发挥作用。有趣的是，OaAEP1b不能在近缘老鹳草环肽前体的N端位点切割，这表明环肽生物合成中还涉及其他蛋白酶。最后，我们通过与环肽无关的肽的环化证明了这种酶的广泛用途。我们提出，重组OaAEP1b是一种强大的工具，可用于需要提高肽产品稳定性的肽工程应用中。
