Investigation of the Lacticin 481 Synthetase and Its Substrate LctA in the Production of the Antibiotic Lacticin 481

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antimicrobial agents produced by Gram-positive bacteria that contain thioether cross-links termed lanthionines. Lantibiotic biosynthesis begins with a precursor peptide (LanA) consisting of leader and propeptide regions. The non-proteinogenic amino acids are introduced by the dehydration of serine and threonine residues in the propeptide followed by stereoselective intramolecular Michael-type addition of cysteines onto the unsaturated amino acids. Class I lantibiotics are biosynthesized by a LanB dehydratase and a LanC cyclase while class II lantibiotics are generated by one bifunctional LanM enzyme. Lacticin 481 synthetase (LctM) catalyzes the post-translational modifications required for the generation of the lantibiotic lacticin 481. Previous studies in our laboratory revealed the protein responsible for the creation of the intricate polycyclic antimicrobial agent was tolerant of several point mutants within its peptide substrate LctA. In an effort to better understand the substrate specificity of LctM such that the enzyme could be utilized to engineer novel therapeutic peptides, further mutational studies were conducted on LctA. Mutation of conserved amino acids within the leader peptide did not abolish LctM activity even though the leader was necessary for full and efficient substrate processing. Studies conducted on the propeptide region uncovered the distance of a serine from the leader peptide and its flanking residues play a role in enzymatic dehydration. Additional propeptide mutants were constructed to examine key structural features of lacticin 481 and their importance in antibiotic activity. Furthermore, the broad specificity of LctM allowed for preparation of other class II lacticin 481 family members including nukacin ISK-1, mutacin II and ruminococcin A. Finally, ICP analysis revealed LctM is a metalloprotein containing one equivalent of zinc. Mutation of the zinc ligands C781 and C836 to alanine in LctM did not affect dehydration activity but did impair cyclization activity of the protein. These results implicate the C-terminal domain of LctM in cyclization, show that the zinc ligands are critical for cyclization, and reveal that dehydration takes place independently from cyclization.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Patton, Gregory

Contributors dc:contributor

• van der Donk, Wilfred A.

• Chemistry, Biochemistry

Identifier

(UMI)AAI3337885

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/72248