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This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
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.
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.
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.
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.
Nostoc punctiforme is a filamentous cyanobacterium capable of forming symbiotic associations with a wide range of plants. The strain exhibits extensive phenotypic characteristics and can differentiate three mutually exclusive cell types: nitrogen-fixing heterocysts, motile hormogonia and spore-like akinetes. Here, we provide evidence for a crucial role of an extracellular metabolite in balancing cellular differentiation. Insertional mutagenesis of a gene of the polyketide synthase gene cluster pks2 led to the accumulation of short filaments carrying mostly terminal heterocysts under diazotrophic conditions. The mutant has a strong tendency to form biofilms on solid surfaces as well as in liquid culture. The pks2-strain keeps forming hormogonia over the entire growth curve and shows an early onset of akinete formation. We could isolate two fractions of the wildtype supernatant that could restore the capability to form long filaments with intercalary heterocysts. Growth of the mutant cells in the neighbourhood of wild-type cells on plates led to a reciprocal influence and a partial reconstruction of wild-type and mutant phenotype respectively. We postulate that extracellular metabolites of Nostoc punctiforme act as life cycle governing factors (LCGFs) and that the ratio between distinct factors may guide the differentiation into different life stages.