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Title:Investigations of the Mechanism and Substrate Scope of the Enzymes Involved in Lantibiotic Biosynthesis
Author(s):Chatterjee, Champak
Doctoral Committee Chair(s):van der Donk, Wilfred A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Abstract:The subtilin prepeptide SpaS consists of 56 amino acids that includes five cysteine residues. This peptide is the substrate of the putative lantibiotic dehydratase, SpaB, and cyclase SpaC. The heterologously expressed and purified SpaS peptide was employed in assays with the purified SpaB and SpaC proteins to investigate conditions for the in vitro reconstitution of these proteins. The successful in vitro reconstitution of the lacticin 481 synthetase, LctM, led to preliminary investigations of its low substrate specificity. In this thesis the substrate specificity of LctM was tested further with non-proteinogenic amino acids. The effect of D-amino acids in the truncated prepeptide, LctA, on LctM action was determined. The ability of LctM to catalyze the cyclization of unusual substrates bearing cysteine analogues such as L-homocysteine was demonstrated. The mechanism of dehydration of Ser/Thr residues by LctM was also interrogated. Truncated substrates containing phosphorylated serine and threonine residues underwent dehydration by Lctm in the absence of ATP when ADP was present in the assays. In conjunction with the identification of phosphorylation in the structural region of two different truncated mutants, these results suggest that LctM may catalyze phosphorylation of Ser/Thr residues in its substrate prior to their dehydration. Mutations of highly conserved residues in the leader sequence as well as deletions of the N-terminal residues in the leader sequence of LctA did not affect dehydration by LctM.
Issue Date:2005
Description:160 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.
Other Identifier(s):(MiAaPQ)AAI3182235
Date Available in IDEALS:2015-09-25
Date Deposited:2005

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