4-Methylproline guided natural product discovery: co-occurrence of 4-hydroxy- and 4-methylprolines in nostoweipeptins and nostopeptolides.

4-methylproline (4-mPro) is a rare nonproteinogenic amino acid produced by cyanobacteria through the action of a zinc-dependent long-chain dehydrogenase and a Δ(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue. Here, we used the presence of 4-mPro biosynthetic genes to discover new bioactive compounds from cyanobacteria. Eight biosynthetic gene clusters containing the 4-mPro biosynthetic genes nosE and nosF were found from publicly available cyanobacteria genomes, showing that 4-mPro is a good marker to discover previously unknown nonribosomal peptides. A combination of polymerase chain reaction (PCR) and liquid chromatography-mass spectroscopy (LC-MS) methods was used to screen 116 cyanobacteria strains from 8 genera. The 4-mPro biosynthetic genes were detected in 30 of the 116 cyanobacteria strains, 12 which were confirmed to produce 4-mPro by amino acid analysis. Species from the genus Nostoc were responsible for 80% of the positive results. Altogether, 11 new nonribosomal cyclic peptides, nostoweipeptin W1-W7 and nostopeptolide L1-L4, were identified from Nostoc sp. XPORK 5A and Nostoc sp. UK2aImI, respectively, and their chemical structure was elucidated. Interestingly, screening with 4-mPro genes resulted in the detection of peptides that do not contain just one 4-mPro but also 4-hydroxylproline (nostopeptolides) and, in case of nostoweipeptins, two 4-mPros and two 4-hydroxyprolines. Peptides from both groups inhibit microcystin-induced apoptosis of hepatocytes HEK293. The cell experiments indicated that these cyclic peptides inhibit the uptake of microcystin by blocking the organic anion-transporters OATP1B1/B3. This study enriches the drug library of microcystin antitoxin.