title: Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries.
abstract: Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.
authors.affiliation: Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24-25, 14476, Potsdam-Golm, Germany.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Reyna-González
authors.affiliation: Institute of Chemistry, Technical University Berlin, Straße des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Schmid
authors.affiliation: Institute of Chemistry, Technical University Berlin, Straße des 17. Juni 124, 10623, Berlin, Germany.|Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0751, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Petras
authors.affiliation: Institute of Chemistry, Technical University Berlin, Straße des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Roderich D
authors.initials: RD
authors.lastname: Süssmuth
authors.affiliation: Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24-25, 14476, Potsdam-Golm, Germany. editt@uni-potsdam.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Dittmann
mesh_terms: D053898:Biosynthetic Pathways; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D020860:Subtilisin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D010456:Peptides, Cyclic; D015842:Serine Proteinase Inhibitors; D014357:Trypsin; D020860:Subtilisin
keywords: biosynthesis; cyanobacteria; microviridins; natural products; peptides