Characterization of glutamyl-tRNA–dependent dehydratases using nonreactive substrate mimics

Abstract

The 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 tRNAGlu to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids. Here, we describe the synthesis of inert analogs that mimic substrate glutamyl-tRNAGlu and the glutamylated peptide intermediate, and determine the crystal structures of 2 LanBs in complex with each of these compounds. Mutational studies were used to characterize the function of the glutamylation and glutamate elimination active-site residues identified through the structural analysis. These combined studies provide insights into the mechanisms of substrate recognition, glutamylation, and glutamate elimination by LanBs to effect a net dehydration reaction of Ser and Thr.

A Structural View on the Maturation of Lanthipeptides

“…Subsequently, the elimination of these activated residues resulted in the dehydrated residues Dha and Dhb. Of course, this reconstitution allowed a detailed study of the catalytic activity and the identification of essential amino acids of this LanB dehydratase (Garg et al, 2013;Bothwell et al, 2019). In 2015, the fruitful collaboration of the Wilfred van der Donk and Satish K. Nair groups also reported the crystal structure of NisB ( Figure 3A).…”

“…Cartoons were generated using PyMol (www.pymol.org). (Bothwell et al, 2019). Thus, the reason for this difference is still an open question.…”

Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity

“…We previously reported the structure of AlbC in complex with a dipeptidyl intermediate, shedding light on aminoacyl binding by CDPSs ( 11 ). In the case of LanBs and L/F transferases, structures have been reported with synthetic analogues of puromycin, which mimic the 3′-terminal aminoacylated adenosines of AA-tRNAs and in which the labile ester bond between the nucleotide and the amino acid is replaced by a stable amide bond ( 48 , 50 ). Using peptidyl-RNA conjugates produced by a solid-phase synthesis/click chemistry approach, Fonvielle et al.…”

Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

“…rapidly expanding with the discovery and characterization of many new biosynthetic pathways, elucidation of the structural features that endow these enzymes with their relaxed substrate specificities and interesting catalytic properties have lagged behind. Nevertheless, high-resolution structures have been solved for a handful of enzymes involved in the biosynthesis of several types of RiPP natural products including the class I and II lanthipeptides, [6][7][8][9][10] cyanobactins, [11][12] orbitides, 13 sactipeptides, 14 lasso peptides, 15 thiopeptides, 16 and others. [17][18][19] In several systems, co-crystal structures of the RiPP biosynthetic enzyme bound to its cognate precursor peptide have revealed the presence of a winged helix-turn-helix motif (termed the RiPP recognition element 20 Recently, we reported the use hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a versatile biophysical tool for studying the structural dynamics of RiPP biosynthetic enzymes.…”