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Title:Biochemical characterization of enzymes involved in the post-translational modification of lantibiotics
Author(s):Ortega, Manuel A
Director of Research:van der Donk, Wilfred A
Doctoral Committee Chair(s):van der Donk, Wilfred A
Doctoral Committee Member(s):Nair, Satish K; Martinis, Susan A; Hergenrother, Paul J
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):antibiotics
biosynthesis
enzymology
X-ray crystallography
biochemistry
tRNA biology
natural products
ribosomal peptide natural products
Ribosomally synthesized and post-translationally modified peptides (RiPPs)
Lantipeptides
Lanthipeptides
Lantibiotics
leader peptide
nisin
microbisporicin
NAI-107
dehydration
thioether
epilancin 15x
halogenation
decarboxylation
proteolysis
combinatorial biosynthesis
post-translational modifications
glutamylation
Abstract:Lantibiotics are ribosomally-synthesized and post-translationally modified peptides (RiPPs) characterized for exerting antimicrobial activity against bacterial strains resistant to commonly used antibiotics. During lantibiotic biosynthesis different enzymes install various post-translational modifications (PTMs) in a precursor peptide important for conferring their biological activity. Understanding how these biosynthetic enzymes catalyze their respective reactions is central to further develop these compounds for therapeutic applications. This dissertation presents the biochemical characterization of different lantibiotic biosynthetic enzymes responsible for the dehydration, decarboxylation, halogenation, and proteolysis of selected lantibiotics. During class I lantibiotic biosynthesis selected Ser/Thr residues in a precursor peptide are dehydrated by a lantibiotic dehydratase. Biochemical and structural studies in collaboration with the Nair Laboratory identified lantibiotic dehydratases to be glutamyl-tRNAGlu-dependent enzymes. The role of glutamyl-tRNAGlu in the dehydration process, the generality of glutamyl-tRNAGlu usage by lantibiotic dehydratases, as well as identity elements within the tRNA needed for recognition by lantibiotic dehydratases were established. A co-crystal structure of a lantibiotic dehydratase in complex with its substrate peptide is discussed, providing the first insights into substrate recognition by these enzymes. In addition to dehydroamino acids, selected lantibiotics may contain additional unusual PTMs such as decarboxylations and halogenations. In this thesis, the substrate specificity of a lantibiotic cysteine decarboxylase and a lantibiotic tryptophan halogenase was characterized using mass spectrometry and bioinformatic approaches. In contrast to many other lantibiotic biosynthetic enzymes, which employ the use of a leader peptide for substrate recognition, both enzymes catalyzed their respective reactions in a leader peptide independent manner. The potential of both enzymes to be used as general tools for modifying non-cognate peptides for various applications was investigated and discussed. Finally, the last step in lantibiotic maturation involves the removal of an N-terminal leader peptide by a lantibiotic peptidase. Bioinformatic, kinetic and mass spectrometry analysis revealed a consensus motif within precursor peptides important for cleavage by these peptidases. The use of this enzyme as a general tool for leader peptide removal from other RiPPs is explored and discussed. Together this dissertation signifies fundamental advances in RiPP biosynthesis with direct implications in the bioengineering of such compounds for clinical uses.
Issue Date:2015-11-10
Type:Thesis
URI:http://hdl.handle.net/2142/89190
Rights Information:Copyright 2015 Manuel A. Ortega
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12


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