4-Methylproline Guided Natural Product Discovery: Co-Occurrence of 4-Hydroxy- and 4-Methylprolines in Nostoweipeptins and Nostopeptolides

Abstract

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.

A Genetic and Chemical Perspective on Symbiotic Recruitment of Cyanobacteria of the Genus Nostoc into the Host Plant Blasia pusilla L.

“…Nostocyclopeptides are structurally similar to nostopeptolides, the gene cluster organization of Ncp and Nos operons share many common features, and there is a high degree of homology between key genes in the operons (Becker et al, 2004; Liu et al, 2014). This may suggest a similar biological function of the two compounds.…”

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium Nostoc punctiforme

“…GSV 224 (18). Related gene clusters were predominantly detected in different Nostoc isolates, including strains isolated from lichen (19). This could be an indication that nostopeptolides (and related peptides) are specifically connected with the Nostoc lifestyle.…”

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria

“…Chemical diversity of nonribosomal peptides and polyketides can be further increased through the supply of unusual precursors or enzymatic tailoring reactions [11,14,16,[19][20][21][22][23][24][25][26]. Table S1).…”