Peptide epimerase-dehydratase complex responsible for biosynthesis of the linaridin class ribosomal peptides.

Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids.

Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene.

Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D-amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D-amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D-amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.

A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe.

We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.