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Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.
Microviridins are unique protease inhibitors from bloom-forming cyanobacteria that have both ecological and pharmacological relevance. Their peptide backbones are produced ribosomally, and ATP grasp ligases introduce omega-ester and omega-amide bonds to yield rare cage-like structures. Bioinformatic analysis of the microviridin biosynthesis gene cluster suggests a novel type of processing machinery, which could rationalize the challenging in vivo/in vitro reconstitution of the pathway. In this work, we report the establishment of a minimal expression system for microviridins. Through bioinformatics and mutational analysis of the MdnA leader peptide we identified and characterized a strictly conserved binding motif that is specific for microviridin ligases. Furthermore, we showed that the ABC transporter MdnE is crucial for cyclization and processing of microviridins and demonstrated that MdnE is essential for stability of the microviridin biosynthesis complex.
Microviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization.
Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions
(2014)
Understanding and controlling proteolysis is an important goal in therapeutic chemistry. Among the natural products specifically inhibiting proteases microviridins are particularly noteworthy. Microviridins are ribosomally produced and posttranslationally modified peptides that are processed into a unique, cagelike architecture. Here, we report a combined rational and random mutagenesis approach that provides fundamental insights into selectivity-conferring moieties of microviridins. The potent variant microviridin J was co-crystallized with trypsin, and for the first time the three-dimensional structure of microviridins was determined and the mode of inhibition revealed.