Anatoxin-a(S) is the most potent natural neurotoxin produced by fresh water cyanobacteria. It is also the least understood and monitored. Although this potent cholinesterase inhibitor was first reported in the 1970s and connected with animal poisonings, the lack of chemical standards and identified biosynthetic genes together with limited diagnostics and acute reactivity of this naturally-occurring organophosphate have limited our understanding of its environmental breadth and human health implications. Anatoxin-a(S) irreversibly inhibits acetylcholinesterase much like other organophosphate agents like paraoxon. It is however often confused with the similarly named anatoxin-a that has a completely different chemical structure, mechanism of action, and biosynthesis. Herein we propose renaming of anatoxin-a(S) to clarify its distinct structure and mechanism and to draw renewed attention to this potent natural poison. We propose the new name guanitoxin (GNT) to emphasize its distinctive guanidino organophosphate chemical structure.
Title: Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants Chekan JR, Estrada P, Covello PS, Nair SK Ref: Proc Natl Acad Sci U S A, 114:6551, 2017 : PubMed
Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.
Title: Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates Chekan JR, Koos JD, Zong C, Maksimov MO, Link AJ, Nair SK Ref: Journal of the American Chemical Society, 138:16452, 2016 : PubMed
Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs), with a threaded knot structure that is formed by an isopeptide bond attaching the N-terminus of the peptide to a side chain carboxylate. Some lasso peptide biosynthetic clusters harbor an enzyme that specifically hydrolyzes the isopeptide bond to yield the linear peptide. We describe here the 2.4 A resolution structure of a lasso peptide isopeptidase revealing a topologically novel didomain architecture consisting of an open beta-propeller appended to an alpha/beta hydrolase domain. The 2.2 A resolution cocrystal structure of an inactive variant in complex with a lasso peptide reveals deformation of the substrate, and reorganization of the enzyme active site, which exposes and orients the isopeptide bond for hydrolysis. Structure-based mutational analysis reveals how this enzyme recognizes the lasso peptide substrate by shape complementarity rather than through sequence specificity. The isopeptidase gene can be used to facilitate genome mining, as a network-based mining strategy queried with this sequence identified 87 putative lasso peptide biosynthetic clusters, 65 of which have not been previously described. Lastly, we validate this mining approach by heterologous expression of two clusters encoded within the genome of Asticcaucalis benevestitus, and demonstrate that both clusters produce lasso peptides.
Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 A), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 A), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 A) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.
