The cyanobacterial hepatotoxin microcystin is assembled at a non-ribosomal peptide synthetase (NRPS) complex. The enormous structural diversity of this peptide, which is also found in closely related strains, is the result of frequent recombination events and point mutations. Here, we have compared the in vitro activation profiles of related monospecific and multispecific modules that either strictly incorporate leucine or arginine or incorporate chemically diverse amino acids in parallel into microcystin. By analyzing di- and tri-domain proteins we have dissected the role of adenylation and condensation domains for substrate specificity. We have further analyzed the role of subdomains and provide evidence for an extended gatekeeping function for the condensation domains of multispecific modules. By reproducing natural point mutations, we could convert a monospecific module into a multispecific module. Our findings may inspire novel synthetic biology approaches and demonstrate how recombination platforms of NRPSs have developed in nature.

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.

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.

The biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation through the carboxylic acid side chains of d-glutamate and β-methyl d-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains, McyE-A and McyB-A , either in a didomain or a tridomain context with the cognate thiolation domain and the upstream condensation domain revealed that substrate activation of both domains strictly depended on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond-forming modules incorporating d-glutamate or d-aspartate. Our findings further contribute to the understanding of the multifaceted role of condensation domains in nonribosomal peptide synthetase assembly lines.

Ein wichtiges Ziel in der therapeutischen Chemie ist das Verständnis und die Kontrolle von proteolytischen Prozessen. Unter den proteaseinhibierenden Naturstoffen nehmen Microviridine eine Sonderstellung ein. Microviridine sind ribosomal erzeugte und posttranslational modifizierte Peptide, die zu einer einzigartigen Struktur mit käfigartiger Gestalt prozessiert werden. Hier beschreiben wir einen kombinierten Ansatz aus rationaler und randomisierter Mutagenese, der grundlegende Erkenntnisse über die Bestandteile der Microviridinstruktur liefert, die zur Selektivität beitragen. Die aktive Variante Microviridin J wurde mit Trypsin kokristallisiert. Dabei wird erstmals die dreidimensionale Struktur von Microviridinen demonstriert und die Art der Inhibierung dargestellt.Hintergrundinformationen zu diesem Beitrag sind im WWW unter http://dx.doi.org/10.1002/ange.201309721 zu finden.