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ERIC Number: ED526757
Record Type: Non-Journal
Publication Date: 2009
Pages: 159
Abstractor: As Provided
Reference Count: 0
ISBN: ISBN-978-1-1095-7496-8
ISSN: N/A
Investigation of the Enzymes Involved in Lantibiotic Biosynthesis: Lacticin 481 and Haloduracin
Ihnken, Leigh Anne Furgerson
ProQuest LLC, Ph.D. Dissertation, University of Illinois at Urbana-Champaign
Lantibiotics are cyclic peptides that exhibit a range of biological properties, including antimicrobial activity. They are ribosomally-synthesized as linear precursor peptides that consist of two regions, an N-terminal leader peptide and a C-terminal propeptide (or structural) region. The structural region undergoes extensive enzyme-catalyzed post-translational modification, while the leader peptide remains unmodified until it is proteolytically removed in the final steps of lantibiotic biosynthesis. The hallmark post-translational modifications of lantibiotics include the dehydroamino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb), from enzymatic dehydration of Ser (Dha) and Thr (Dhb), and the macrocyclic thioether crosslinks termed lanthionine (Lan, from reaction with Dha) and methyllanthionine (MeLan, from reaction with Dhb) rings that arise from Michael-like addition of Cys thiols to the unsaturated amino acids. Following structural region modification, the leader peptide is removed and the mature lantibiotic is exported outside the cell. All three processes leading to the generation of a mature lantibiotic (dehydration, cyclization, and proteolysis) were investigated and are reported in this thesis. LctM is the lacticin 481 synthetase that catalyzes dehydration of Ser/Thr residues in the lacticin 481 precursor peptide LctA. Dehydration occurs via phosphopeptide intermediates in which the Ser/Thr side chain hydroxyl groups are phosphorylated with the [gamma]-phosphate of ATP. A subsequent phosphate elimination step gives rise to the [alpha], [beta]-unsaturated residues Dha and Dhb. A variant of LctM in which Thr405 was mutated to Ala exhibited impaired elimination activity and was utilized, in this study, as a general Ser/Thr kinase to effect phosphorylation of these residues in a random peptide library. Other uses of ATP for LctM and the haloduracin beta synthetase HalM2, were explored using [alpha]- and [gamma]-[superscript32] P ATP radioassays. These studies indicated the LanM proteins do not use autophosphorylation as a means of autoactivation, and they do not stably phosphorylate their cognate LanA substrates at positions other than those targeted for dehydration. Further exploration of the LanM dehydration mechanism using high-resolution tandem mass spectrometry demonstrated that the dehydration reaction proceeded in a distributive and directional fashion, from N- to C- terminus. The thiol-alkylation reaction that gives rise to (Me)Lan rings present in mature lantibiotics is also catalyzed by LanM synthetases in lacticin 481 and haloduracin beta biogenesis. The C-terminus of these large bifunctional enzymes contains a catalytic zinc atom that is important for cyclization activity. The zinc ligands for HalM2 were identified and experiments with cobalt-reconstituted HalM2 supported a mechanism for (Me)Lan cyclization involving a metal-coordinated substrate thiolate intermediate. Furthermore, HalM2-catalyzed cyclization was distributive and proceeded in a directional manner from N- to C-terminus, as determined by examination of reaction intermediates by high-resolution tandem mass spectrometry. Finally, removal of the leader peptide from the modified precursor peptides is the final step in the maturation of lantibiotics, and is required for achievement of full biological activity. In lacticin 481 maturation, this peptide cleavage reaction is performed by the N-terminal protease domain of the lacticin 481 transporter, LctT. The activity of the protease domain of LctT was reconstituted "in vitro" and its substrate specificity was probed with numerous variants of the lacticin 481 precursor peptide. The N-terminal 150 amino acids of LctT were able to process most LctA mutant peptides, with the exception of peptides containing mutations at the double glycine motif located at the junction between leader sequence and structural region. Cys protease activity was confirmed for LctT as mutation of Cys12 to Ser or Ala abolished protease activity. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.]
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Publication Type: Dissertations/Theses - Doctoral Dissertations
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A