Investigations of peptide aminoacyl tRNA ligase (PEARL) biosynthetic gene clusters

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Daniels, Page Nicole

Contributors dc:contributor

• van der Donk, Wilfred A
• Hergenrother, Paul J
• Nair, Satish K
• Tajkhorshid, Emad

• PEARLs
• RIPPs
• natural products
• enzymology
• biosynthesis

Statement dc:rights

• Copyright 2022 Page Daniels

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/117525

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F117525