Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

Abstract

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.Ribosomally synthesized and post-translationally modified peptides (RiPPs) 3 (1) are a diverse set of natural products that are formed by the action of maturation enzymes on a linear peptide substrate. An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2-8). Lasso peptides are a class of RiPPs, characterized by a threaded structure resembling a slipknot (9 -11). The internal macrocycle is realized via a single isopeptide bond installed post-translationally by two maturation enzymes (12). While there is a quickly expanding literature about the lasso peptide maturation enzymes (12-14), there is little known about the catabolism of these molecules. Recently, we reported an enzyme, lasso peptide isopeptidase, that specifically hydrolyzes the isopeptide bond of lasso peptides (Fig. 1A) (15). This enzyme, related to prolyl oligopeptidases, was found in the vicinity of a lasso peptide gene cluster in the freshwater ␣-proteobacterium Asticcacaulis excentricus. This organism has two separate lasso peptide gene clusters, each with an associated isopeptidase gene (15, 16). There have been a large number of lasso peptides discovered in proteobacteria (11,17), and an isopeptidase is commonly associated with such clusters (9).We named the two isopeptidases in A. excentricus AtxE1 and AtxE2. The gene for AtxE1 is found next to the gene cluster that encodes for the biosynthesis of astexin-1, while the AtxE2 gene is located next to the gene cluster encoding astexins-2 and -3 (Fig. 1B) (15). We have previously characterized AtxE2 in vitro (15). This enzyme cleaves astexin-2 and astexin-3, but no crossreactivity was observed between AtxE2 and astexin-1. Whereas astexin-2 and astexin-3 share relatively high sequence homology (identity at 13/24 positions), the astexin-1 sequence is more divergent (Fig. 1C). In addition, we have shown that, at least for astexin-2, the thermally unthreaded variant of the peptide is not a substrate for Atx...

Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates

“…The Ser10-Gln14 isopeptide loop, which is converted from a linear to a loop topology during lasso maturation, is proposed to play a role in the biosynthetic process. 8 Our studies indicate this loop also plays an important function in the catabolism of lasso peptides, as binding to the isopeptidase repositions this loop away from the isopeptide bond and into a binding pocket. Hence, it is likely that both the RiPP biosynthetic and catabolic enzymes utilize the same epitope in the substrate for recognition.…”

“…This large pocket has a volume of 8988 Å 3 as measured by CASTp using a probe size of 2.5 Å. 13 This would easily accommodate the astexin-3 lasso peptide substrate 8 , which as a volume of 2457 Å 3 as calculated by Chimera. 14 …”

“…The absence of extensive side-chain specific interactions explains the tolerance AtxE2 for the panel of astexin-3 Ala variants, almost all of which are substrates for the enzyme. 8 …”

“…23 The broad tolerance of the isopeptidase likely is necessary to allow for hydrolysis of point mutants that may occur in the substrate, which is also gene encoded. 8 …”

Lasso Peptide Benenodin-1 Is a Thermally Actuated [1]Rotaxane Switch

“…11 The isopeptidase only cleaves threaded substrates, accommodating the “loop” region of the rotaxane thread in a cavity within the enzyme active site. 12–13 …”