Mechanistic Studies of Lantibiotic Biosynthetic Enzymes and Discovery of Novel Lanthionine-Containing Peptides
Li, Bo
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https://hdl.handle.net/2142/72341
Description
Title
Mechanistic Studies of Lantibiotic Biosynthetic Enzymes and Discovery of Novel Lanthionine-Containing Peptides
Author(s)
Li, Bo
Issue Date
2009
Doctoral Committee Chair(s)
van der Donk, Wilfred A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Lantibiotics are ribosomally synthesized antimicrobial peptides containing unique thioether crosslinks, lanthionines and methyllanthionines. These motifs are introduced posttranslationally in a two-step process, which involves the dehydration of serines and threonines into dehydroalanines and dehydrobutyrines, respectively, and subsequent intramolecular Michael-type additions of cysteines to the dehydrated amino acids. The dehydration and cyclization are either catalyzed by two separate enzymes, a dehydratase LanB and a cyclase LanC, or a single bifunctional enzyme LanM. Here we demonstrated the first in vitro reconstitution of the activity of a LanC cyclase, NisC, which is involved in the cyclization process of nisin, the prototypic lantibiotic widely used as a food preservative. The X-ray crystal structure of NisC revealed the presence of a zinc ion and provided insights into the molecular mechanism of the cyclization reaction. Mutagenesis of residues conserved among the LanC family of proteins and located in the zinc site of the NisC structure indicated that the three zinc ligands and conserved residues His212 and Asp141 were essential for the activity of NisC. His212 was proposed to serve as the catalytic acid or base in the cyclization reaction. In addition to the mechanistic studies of the NisC cyclase, we present here the genome-enabled discovery of a group of novel lanthionine-containing peptides, termed prochlorosins, which are produced by planktonic marine cyanobacteria Prochlorococcus MIT9313. This strain was shown to utilize a single LanM enzyme to generate as many as 16 conformationally constrained peptides of remarkably diverse structures, representing an impressive case of natural combinatorial biosynthesis. The thioether ring topologies were determined for several of the prochlorosins. Finally, we demonstrate the initial characterization of a LanM-like enzyme containing a serine/threonine protein kinase domain, and describe our efforts in the in vitro reconstitution of the LanB family of dehydratases from several bacterial strains and a halogenase involved in tryptophan chlorination.
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