Maksimov Mikhail O, Koos Joseph D, Zong Chuhan, Lisko Bozhena, Link A James
From the Departments of Chemical and Biological Engineering.
Molecular Biology, and.
J Biol Chem. 2015 Dec 25;290(52):30806-12. doi: 10.1074/jbc.M115.694083. Epub 2015 Nov 3.
Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.
套索肽异肽酶是一种特异性水解套索肽异肽键，使这些肽呈线性的酶。为了对来自偏心柄杆菌的套索肽异肽酶AtxE2进行详细的构效分析，我们解析了其底物astexin-2和astexin-3的核磁共振结构。通过体外酶活性测定，我们发现这些肽的C末端尾部对于异肽酶活性而言是可有可无的。构建了一系列在环和环内每个位置具有丙氨酸取代的astexin-2和astexin-3变体，并且我们发现除了一个之外，所有这些肽都被异肽酶切割。因此，与套索肽生物合成酶非常相似，套索肽异肽酶具有广泛的底物特异性。对切割反应的定量分析表明，这些肽环位置的丙氨酸取代导致切割减少，这表明环作为异肽酶的识别元件。
