Biosynthesis of the antimicrobial peptide epilancin 15X

Velasquez, Juan

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https://hdl.handle.net/2142/29545 Copy

Description

Title

Biosynthesis of the antimicrobial peptide epilancin 15X

Author(s)

Velasquez, Juan

Issue Date

2012-02-01T00:54:48Z

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

van der Donk, Wilfred A.

Committee Member(s)

• Metcalf, William W.
• Oldfield, Eric
• Silverman, Scott K.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Antibiotics
• Lantibiotics
• Epilancin 15X
• Nisin
• Antimicrobial peptides
• Biosynthesis
• Natural Products
• Short-Chain Dehydrogenase/Reductase
• Serine proteases
• Phosphonates

Abstract

Lantibiotics are polycyclic antimicrobial peptides that are ribosomally synthesized and posttranslationally modified to their biologically active forms. The recently discovered lantibiotic epilancin 15X produced by Staphylococcus epidermidis 15X154 is active against several pathogenic bacteria, including methicillin-resistant S. aureus and vancomycin-resistant Enterococci. Epilancin 15X contains an unusual N-terminal lactate group (Lac) that could be important for its bioactivity. To understand its biosynthesis, the epilancin 15X gene cluster was identified. The Lac group is introduced by dehydration of a Ser residue in the first position of the core peptide by a lantibiotic dehydratase (ElxB), followed by proteolytic removal of the leader peptide by a serine-type protease (ElxP), and hydrolysis of the resulting N-terminal dehydroalanine residue. A pyruvyl group thus formed is finally reduced to Lac by a dehydrogenase (ElxO). Using substrate analogs synthesized by solid-phase peptide synthesis or heterologously expressed in Escherichia coli, the enzymatic activities of ElxO and ElxP and their substrate specificities were investigated in vitro. Furthermore, the dehydratase activity of ElxB was demonstrated in vivo. ElxO tolerates several structural modifications along the substrate sequence and can catalyze the NADPH-dependent reduction of diverse pyruvyl- and oxobutyryl-containing peptides, such as the lantibiotic lactocin S, to the corresponding alcohols. The reaction mechanism of ElxO was proposed based on its x ray crystal structure and a mutagenesis study. The characterization of ElxP demonstrated that the protease can cleave the unmodified precursor peptide and relies on the C-terminal residues of the leader region for substrate recognition. Finally, the stereochemical configuration and the importance of the N-terminal Lac for peptide stability against bacterial aminopeptidases were determined. Understanding the biosynthetic route to epilancin 15X opens the door for engineering of novel lantibiotics with more potent biological activities or different spectra of action.

Graduation Semester

2011-12

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http://hdl.handle.net/2142/29545

Copyright and License Information

Copyright 2011 Juan Esteban Velásquez

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