Use of a scaffold peptide in the biosynthesis of amino acid–derived natural products

Abstract

Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA–dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid–derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid–derived natural products.

Natural products in drug discovery: advances and opportunities

“…The progressive development of higher-field NMR instruments and probe technology 60 , 61 has enabled NP structure determination from very small quantities (below 10 µg) 62 , 63 , which is important, as the available quantities of NPs are often limited. In addition, microcrystal electron diffraction (MicroED) has recently emerged as a cryo-electron microscopy-based technique for unambiguous structure determination of small molecules 64 and is already finding important applications in NP research 65 . The increased resolution and sensitivity of analytical equipment can also help address problems associated with ‘residual complexity’ of isolated NPs; that is when biologically potent but unidentified impurities in an isolated NP sample (which could include structurally related metabolites or conformers) lead to an incorrect assignment of structure and/or activity 66 , 67 .…”

MicroED with the Falcon III direct electron detector

“…Because crystals are screened and imaged using the same optical elements that are ultimately used to collect diffraction data, the large magnification of a transmission electron microscope (TEM) can be leveraged to select crystals with side lengths of as small as 50 nm (Rodriguez et al, 2015). In contrast to single-particle cryo-EM, crystal constraints provide near-perfect alignment of the molecules and therefore the measured signal is strong enough to yield high-resolution structural information even from small peptides or chemical compounds (Gallagher-Jones et al, 2018; Jones et al, 2018; Ting et al, 2019). MicroED thus provides a means to determine structures that are not attainable by other methods.…”

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

“…Together, these advances have led to the discovery of numerous new natural products [24][25][26][27][28] and, in the case of RiPPs, new post-translational modifications. 14,[29][30][31][32][33][34][35] Bioinformatics recently has been utilized to discover new RiPP classes, such as the α-keto β-amino acid-containing peptides, 36 RiPPs generating thiaglutamate by peptide-amino acyl tRNA ligases (PEARLs), 33 aliphatic ether-containing rotapeptides 31 and non-α thioether-containing ranthipeptides, 37 as well as the pyritides, described herein. Bioinformatics offers a means to prioritize the characterization of divergent BGCs that give rise to novel compounds while also mitigating the historical issue of rediscovery.…”