Structural and mechanistic studies of class III lanthipeptide biosynthesis, an α-N methyltransferase, and a sabath yerba mate methyltransferase

Described in this document is my doctoral thesis work towards the structural and biochemical characterization of proteins involved in bacterial and plant biosynthesis of secondary metabolites. Ribosomally synthetized and post translationally modified peptides (RiPPs) are an important class of natural products. The precursor for all RiPPs is a precursor peptide that can be divided into 2 segments: a leader peptide at the N-terminus and a core peptide at the C-terminus. The leader is typically recognized by tailoring enzymes, and modifications are installed upon the core. For full maturation of a RiPP natural product, the leader peptide is removed by a bacterial protease and exported by a transporter outside of the cell. From this class of natural products, lanthipeptides are characterized by thioether-cross links bis amino acids lanthionine (Lan) and methyllanthionine. (MeLan), and Labionin (Lab) found exclusively produced by class III lanthionine synthetase clusters. In the first chapter, I will describe the in vivo and in vitro step wise reconstitution in E. coli of andalusicin, a class III lantibiotic from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. In the second chapter of this work, I will describe the first crystal structures of a full class III lanthipeptide synthetase for the andalusicin biosynthetic gene cluster: ThurKC, consisting of the lyase, kinase and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structures show interactions between all three domain that results in an active conformation of the kinase domain. My biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provides a framework for engineering of variants of biotechnological interest. In the third chapter, I report the first structure for an α-N methyltransferase of a lanthipeptide BGC (ThurMT) and describe my efforts toward elucidating its substrate scope and specificity. We obtained the NMR structure of one of its active substrates ∆C8 mcThurA1, the first 14 residues of andalusicin modified by ThurKC, that displays an unexpected helical turn in residues Dhb3-Dhb8. I describe the results of our docking and fitting of this peptide to into the active site guided by partial electron density observed in X-ray crystallography datasets, and our assays with point mutants of ThurMT active site cavity to determine importance to catalysis. Finally, I compare this an α-N methyltransferase to other found in RiPP biosynthesis through structural approaches and bioinformatics. Caffeine is the most widely consumed stimulant used by humans, and produced by many plants. The focus of the fourth chapter is the convergently evolved pathway to caffeine biosynthesis found in yerba mate, elucidated by whole genome sequencing of this plant. We identified the three replicates of CS genes that encode the three IpCS SABATH methyltransferases in the in the yerba mate caffeine biosynthetic pathway. I obtained the crystal structure of the last enzyme in this pathway, IpCS3 bound in complex to SAH and its product caffeine. This structure allowed for validation of Alphafold models of IpCS1 and IpCS2 bound to their respective subjects, Xanthine and 3-methylxanthine. Finally, I obtained the characterized the reactivity of IpCS3 with its preferred substrate, theobromine, and paraxanthine. In the appendix of this work, I will enlist the crystallography methods and X-ray crystallographic tables for the structures of eight Influenza (H3N2) neuraminidase proteins, an (H5N2) neuraminidase structure, and three Influenza (H3N2) Hemagglutinin structures that aided the study of the of the evolutionary potential of influenza H3N2 neuraminidases and the effects of such mutants in a sequence-structure-function analysis.

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

2023

Author dc:creator

• Hernandez Garcia, Andrea

Contributors dc:contributor

• Nair, Satish K
• van der Donk, Wilfred A
• Mitchell, Douglas A
• Wu, Nicholas

• Lanthipeptide
• Natural Products
• Structure

Statement dc:rights

• Copyright 2023 Andrea Hernandez Garcia

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/121218

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F121218