From Peptide Precursors to Lanthionine -Containing Peptide Antibiotics Using the First Isolated and Active Lacticin 481 Synthetase: Mechanistic Studies and Peptide Engineering
Xie, Lili
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Permalink
https://hdl.handle.net/2142/84122
Description
Title
From Peptide Precursors to Lanthionine -Containing Peptide Antibiotics Using the First Isolated and Active Lacticin 481 Synthetase: Mechanistic Studies and Peptide Engineering
Author(s)
Xie, Lili
Issue Date
2003
Doctoral Committee Chair(s)
van der Donk, Wilfred A.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
The putative dehydratase SpaB involved in the biosynthesis of subtilin was heterologously overexpressed in E. coli and purified by metal-chelated affinity chromatography. However, in vitro reconstitution using the purified SpaB, SpaC, and the substrate SpaS was unsuccessful. As a result, the biosynthesis of lacticin 481 which employs one single modification enzyme presumably responsible for both dehydration and cyclization was investigated. Both the modification enzyme LctM and the substrate LctA were cloned and purified to homogeneity. The in vitro reconstitution of LctM, the lacticin 481 synthetase, was successful. The assay product was characterized in detail through tandem mass spectrometry, suggesting the formation of identical structural motifs found in lacticin 481. Therefore, LctM is indeed a bifunctional protein, performing both dehydration and cyclization reactions. This study presents the very first in vitro biosynthetic system of any lantibiotic. In addition, upon treatment with the protease Lys-C, the modified peptide in the absence of the leader sequence and the first Lys residue showed biological activity. Mechanistic studies with the active LctM disclosed that the activity of LctM absolutely requires both Mg2+ and ATP. The products of ATP were determined to be ADP and phosphate. The enzyme has low substrate specificity as a series of LctA mutants proved substrates for LctM. Furthermore, peptide engineering was performed, and a large number of lacticin 481 analogs were generated. From these studies, additional structural and mechanistic insights have been elaborated.
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