Mechanistic Investigations of Cyclase Enzymes Involved in Lantibiotic Biosynthesis
Paul, Moushumi
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Permalink
https://hdl.handle.net/2142/84177
Description
Title
Mechanistic Investigations of Cyclase Enzymes Involved in Lantibiotic Biosynthesis
Author(s)
Paul, Moushumi
Issue Date
2005
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)
Chemistry, Biochemistry
Language
eng
Abstract
Lantibiotics are peptide antibiotics that contain thioether bridges, termed lanthionines, putatively generated by Michael addition of cysteines to dehydrated serine and threonine residues in the pre-lantibiotic peptide. In class AI lantibiotics, the post-translational modifications are proposed to involve a multi-enzyme complex including the proteins LanB and LanC that are putatively attributed the functions of dehydration and cyclization, respectively, while class AII use a single enzyme, LanM, to carry out both transformations. Previous work in our lab has shown that the LanC enzymes involved in both subtilin and nisin biosynthesis, SpaC and NisC, respectively, contain stoichiometric amounts of zinc, implying a possible catalytic role. EXAFS analysis of SpaC suggests that two cysteines coordinate to the metal and possibly two histidines. The metal may function to activate the cysteine thiol of the substrate toward intramolecular Michael addition to the dehydroalanine and dehydrobutyrine residues in the peptide. Sequence alignments of both the LanC and LanM families of enzymes reveal four conserved residues, two cysteines and two histidines. Mutants of SpaC replacing the conserved cysteines with alanines were generated and characterization by both EXAFS and ICP analysis has shown that these cysteines are involved in zinc binding. Similar studies have been performed using LctM, the LanM protein involved in lacticin 481 biosynthesis. Cysteine and histidine mutants have been generated and analyzed to determine if eliminating the protein's ability to bind zinc leads to a loss in cyclization activity. Results from these experiments implicate Cys836 and His725 in the cyclization mechanism, and yielded mutant enzymes only capable of dehydrating the LctA substrate. In an attempt to understand the structural requirements for synthetase activity, chimeras consisting of the LctA leader sequence fused to structural regions of various class AI and AII lantibiotics have also been generated and treated with LctM to test for activity.
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