1. Cypemycin Decarboxylase CypD Is Not Responsible for Aminovinyl-Cysteine (AviCys) Ring Formation
Wei Ding, Ning Yuan, Dhanaraju Mandalapu, Tianlu Mo, Suwei Dong, Qi Zhang Org Lett. 2018 Dec 7;20(23):7670-7673. doi: 10.1021/acs.orglett.8b03380. Epub 2018 Nov 19.
The cypemycin decarboxylase CypD is investigated by using a synthetic oligopeptide, which contains the to-be-cyclized dehydroalanine (Dha) residue. It was shown that CypD efficiently catalyzes the decarboxylation of this Dha-containing peptide, but the expected AviCys ring is not formed in the product, suggesting that CypD alone is not enough to form the AviCys ring. It was also shown that the Dha-containing peptide is a better substrate than two similar peptides with a Ser or a Cys residue, supporting that, in cypemycin biosynthesis, Dha formation is prior to decarboxylation of the C-terminal Cys.
2. Movements of the Substrate-Binding Clamp of Cypemycin Decarboxylase CypD
Lei Liu, Stephen Chan, Tianlu Mo, Wei Ding, Shaoning Yu, Qi Zhang, Shuguang Yuan J Chem Inf Model. 2019 Jun 24;59(6):2924-2929. doi: 10.1021/acs.jcim.9b00156. Epub 2019 Apr 29.
Linaridins are a small but growing class of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The class A linaridins, exemplified by cypemycin, possess an unusual S-[( Z)-2-aminovinyl]-d-cysteine (AviCys) residue. Formation of the AviCys in cypemycin requires an oxidative decarboxylation of the precursor peptide C-terminal Cys, and this reaction is catalyzed by a flavin-dependent decarboxylase CypD. In this work, we investigate the molecular recognition processes of CypD by a combination of computational and biochemical analysis. We show that the substrate binding clamp of CypD undergoes dramatic fluctuation, mediating both the substrate entrance into and product release from the catalytic pocket. Extensive molecular dynamic simulations and Fourier transform IR analyses indicated that binding of the substrate induces substantial structural change of the enzyme, converting the substrate-binding clamp from a random loop to a more ordered structure comprising two β sheets and a β turn. The salt bridge between Arg159 guanine and the Cys carboxylate of substrate plays an important role in mediating substrate binding, while hydrophobic interactions are also important in this process. These results provide important mechanistic insights into CypD and other flavin-dependent Cys decarboxylases, and could facilitate future biosynthetic and bioengineering efforts in studying AviCys-containing RiPPs.
3. Substrate specificity of the cypemycin decarboxylase CypD
Wei Ding, Tianlu Mo, Dhanaraju Mandalapu, Qi Zhang Synth Syst Biotechnol. 2018 Sep 15;3(3):159-162. doi: 10.1016/j.synbio.2018.09.002. eCollection 2018 Sep.
The linaridin antibiotic cypemycin is a ribosomal synthesized and post-translationally modified peptide (RiPP) that possesses potent activity against mouse leukemia cells. This peptide natural product contains an S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) moiety in the C-terminus. Formation of AviCys moiety requires an oxidative decarboxylation of the C-terminal Cys of the precursor peptide CypA, and this process is catalyzed by a flavin-containing protein CypD. In this work, we tested CypD substrate specificity with a series of synthetic oligopeptides. We show that most of the N-terminal sequence of CypA is not required for CypD activity, and the C-terminal three residues serve as the minimal structural element for enzyme recognition. We also show that CypD tolerates various substrates with modified C-termini, allowing for the generation of four novel cypemycin variants with modified AviCys moiety by site direct mutagenesis of the precursor peptide CypA. Our study demonstrates the relaxed substrate specificity of CypD and lays a foundation for future bioengineering of AviCys-containing natural products.
