Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%.
View Article and Find Full Text PDFLanthipeptides are ribosomally synthesized and post-translationally modified peptide natural products characterized by the presence of lanthionine and methyllanthionine cross-linked amino acids formed by dehydration of Ser/Thr residues followed by conjugate addition of Cys to the resulting dehydroamino acids. Class I lanthipeptide dehydratases utilize glutamyl-tRNA as a cosubstrate to glutamylate Ser/Thr followed by glutamate elimination. A vast majority of lanthipeptides identified from class I synthase systems have been from Gram-positive bacteria.
View Article and Find Full Text PDFLanthipeptides constitute a major family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are classified into four subfamilies, based on the characteristics of their lanthipeptide synthetases. While over a hundred lanthipeptides have been discovered to date, very few of them are class IV lanthipeptides and the latter are all structurally similar.
View Article and Find Full Text PDFLanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an -methyltransferase that is a member of the most prominent auxiliary enzyme family associated with class I lanthipeptide biosynthetic gene clusters.
View Article and Find Full Text PDFThe peptide natural product nisin has been used as a food preservative for 6 decades with minimal development of resistance. Nisin contains the unusual amino acids dehydroalanine and dehydrobutyrine, which are posttranslationally installed by class I lanthipeptide dehydratases (LanBs) on a linear peptide substrate through an unusual glutamyl-tRNA-dependent dehydration of Ser and Thr. To date, little is known about how LanBs catalyze the transfer of glutamate from charged tRNA to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids.
View Article and Find Full Text PDFCyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles.
View Article and Find Full Text PDFRNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N-allyladenosine (aA) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. aA behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells.
View Article and Find Full Text PDFProtein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally.
View Article and Find Full Text PDFS-adenosyl-L-methionine (SAM) analogues have previously demonstrated their utility as chemical reporters of methyltransferases. Here we describe the facile, large-scale synthesis of Se-alkyl Se-adenosyl-L-selenomethionine (SeAM) analogues and their precursor, Se-adenosyl-L-selenohomocysteine (SeAH). Comparison of SeAM analogues with their equivalent SAM analogues suggests that sulfonium-to-selenonium substitution can enhance their compatibility with certain protein methyltransferases, favoring otherwise less reactive SAM analogues.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
October 2013
Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits.
View Article and Find Full Text PDFA strategy for introducing structural diversity into polyketides by exploiting the promiscuity of an in-line methyltransferase domain in a multidomain polyketide synthase is reported. In vitro investigations using the highly-reducing fungal polyketide synthase CazF revealed that its methyltransferase domain accepts the nonnatural cofactor propargylic Se-adenosyl-l-methionine and can transfer the propargyl moiety onto its growing polyketide chain. This propargylated polyketide product can then be further chain-extended and cyclized to form propargyl-α pyrone or be processed fully into the alkyne-containing 4'-propargyl-chaetoviridin A.
View Article and Find Full Text PDFCurr Protoc Chem Biol
March 2015
Enzymatic transmethylation from the cofactor S-adenosyl-L-methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne-containing SAM mimic, Se-adenosyl-L-selenomethionine (ProSeAM), and a cleavable azido-azo-biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM.
View Article and Find Full Text PDFPosttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases.
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