2026-09-23T10:35:19Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/117525 2025-11-07 col_2142_5131 col_2142_14795 com_2142_5130 com_2142_14794 com_2142_14793 com_2142_8903

van der Donk, Wilfred A van der Donk, Wilfred A Hergenrother, Paul J Nair, Satish K Tajkhorshid, Emad 2022-12 application/pdf en text Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2024-12-01 The student, Page Daniels, accepted the attached license on 2022-08-22 at 13:50. The student, Page Daniels, submitted this Dissertation for approval on 2022-08-22 at 13:57. This Dissertation was approved for publication on 2022-08-30 at 10:52. DSpace SAF Submission Ingestion Package generated from Vireo submission #18467 on 2023-04-12 at 11:34:18 Investigations of peptide aminoacyl tRNA ligase (PEARL) biosynthetic gene clusters Daniels, Page Nicole 2022-08-30 PEARLs RIPPs natural products enzymology biosynthesis Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large, structurally diverse group of natural products that share a common biosynthetic logic. During the biosynthesis of RiPP natural products, a genetically encoded precursor peptide is post-translationally modified by enzymes within the biosynthetic gene cluster. The precursor peptide typically comprises an N-terminal leader peptide and a C-terminal core peptide. The leader peptide serves as a recognition motif for the tailoring enzymes that modify the core peptide. Lanthipeptides are a subset of RiPPs denoted by the presence of lanthionine and methyllanthionine residues. There are currently five classes of lanthipeptides, characterized by their unique biosynthetic machinery. The bioinformatic studies on class I lanthipeptides led to the discovery of a novel class of enzymes called PEARLs or peptide aminoacyl tRNA ligases. PEARLs have low sequence similarity to the N-terminal domain of LanB dehydratases (type I) involved in lanthipeptide biosynthesis. The N-terminal domain of LanBs utilize glutamyl-tRNA to glutamylate the side-chain hydroxyl groups of Ser and Thr residues in a substrate peptide followed by elimination of the Glu by the C-terminal domain to generate dehydroalanine and dehydrobutyrine, but PEARL enzymes lack this elimination domain. PEARLs also use aminoacyl-tRNA as a substrate but perform very different chemistry that first involves ATP-dependent phosphorylation of the C-terminus of a scaffold peptide, and then amide bond formation with the amino group of the aminoacyl-tRNA. This process is involved in the biosynthesis of a range of natural products by phylogenetically diverse bacteria, including the ammosamides and 3-thiaglutamate. Here, two biosynthetic pathways in Alkalihalobacillus halodurans C-125 and Streptomyces sp. CNR-698 that utilize PEARLs were investigated. It was determined that glycyl-tRNA is utilized to install aromatic amines in PEARL-associated pyrroloiminoquinone-type natural products. PEARLs and other biosynthetic enzymes involved in the installation of aromatic amines in these two biosynthetic gene clusters were identified and characterized. A non-PEARL enzyme from the Alkalihalobacillus halodurans C-125 PEARL biosynthetic pathway, BhaC1 that tri-hydroxylates the indole of Trp was probed for its substrate specificity. AlphaFold and AlphaFold Multimer models of BhaC1 and AmmC1 (from Streptomyces sp. CNR-698) help to explain the substrate specificity and suggest a common binding motif. Lastly, the capture and heterologous expression of two biosynthetic gene clusters in Bacillus was investigated: the bha cluster from Alkalihalobacillus halodurans C-125 and the lacticin 481 cluster from Lactococcus lactis. The capture and expression of each cluster was examined to achieve distinct goals: produce and determine the final natural product for the bha cluster and establish a lanthipeptide production system in Bacillus for the lacticin 481 cluster. Appendix A explores the bioconjugation utility of the BhaC1 product. While the product is amenable to conjugation, it is too reactive for our purposes. Thesis eng https://hdl.handle.net/2142/117525 Copyright 2022 Page Daniels Ph.D. Dissertation Biochemistry University of Illinois at Urbana-Champaign Biochemistry