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

Daniels, Page Nicole

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

Description

Title

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

Author(s)

Daniels, Page Nicole

Issue Date

2022-08-30

Director of Research (if dissertation) or Advisor (if thesis)

van der Donk, Wilfred A

Doctoral Committee Chair(s)

van der Donk, Wilfred A

Committee Member(s)

• Hergenrother, Paul J
• Nair, Satish K
• Tajkhorshid, Emad

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• PEARLs
• RIPPs
• natural products
• enzymology
• biosynthesis

Language

eng

Abstract

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.

Graduation Semester

2022-12

Type of Resource

Thesis

Handle URL

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

Copyright and License Information

Copyright 2022 Page Daniels

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