Files in this item



application/pdfYU-DISSERTATION-2015.pdf (8MB)Restricted Access
(no description provided)PDF


Title:Mechanistic studies of class II lanthipeptide synthetases and yeast surface display of lanthipeptide leader peptides
Author(s):Yu, Yi
Director of Research:van der Donk, Wilfred A
Doctoral Committee Chair(s):van der Donk, Wilfred A
Doctoral Committee Member(s):Kranz, David M; Gerlt, John A; Hergenrother, Paul J
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Lanthionine synthetase
Yeast surface display
Abstract:Lanthipeptides are natural products that belong to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). They contain the characteristic lanthionine (Lan) or methyllanthionine (MeLan) structures that contribute to their diverse biological activities. ProcM is a promiscuous bifunctional synthetase that catalyzes both dehydration and cyclization of the lanthipeptides. Its 30 substrate peptides (ProcAs) have a high level of conservation in the N-terminal leader region and hypervariability in the C-terminal core region. A chimeric peptide with a ProcA leader peptide and LctA (the precursor peptide for lacticin 481) core peptide was constructed and co-expressed with ProcM in Escherichia coli. ProcM dehydrated this chimeric peptide up to 5-fold (WT lacticin 481 is dehydrated 4-fold), and also catalyzed the formation of 2~3 rings (WT lacticin 481 has 3 rings). The modified peptide was digested with protease to remove the leader peptide, and the resulting product was demonstrated to have antimicrobial activity against Lactococcus lactis HP. These results showed the promiscuity of ProcM and suggest it may be used to generate cyclic peptide libraries from substrates with a ProcA leader peptide. The product specificity and the regulation of product formation of ProcM were also studied. Despite its structurally very diverse substrate set, high selectivity of product formation from each substrate was observed. The effects of mutation of the conserved residues in the active site, including three zinc ligands (Cys924, Cys970, and Cys971), the proposed active site acid/base His859, and Asp804 that is hydrogen-bonded to His859 were investigated. Mutation of the zinc ligands to alanines or the unique zinc ligand Cys971 to histidine resulted in a decrease of the cyclization rate, especially for the second cyclization of ProcA1.1, ProcA2.8 and ProcA3.3. In the case of ProcA3.3, these mutations also altered the regioselectivity of ProcM and generated a new major product. The H859A mutation completely deactivated the cyclization activity of the enzyme, while the D804N mutation also decreased the cyclization rate but maintained the initial regioselectivity for ProcA3.3. ProcM was not able to correct the ring topology of incorrectly cyclized intermediates and products, suggesting that thermodynamic control is not operational. Instead, the high regioselectivity of product formation appears to be governed by the selectivity of the initially formed ring. By studying truncated enzymes of ProcM, LctM, and CylM, I showed that the N-terminus of these LanMs catalyzed the dehydration and the C-terminal domain catalyzed the cyclization. These two domains were active on their own, indicating that they are relatively independent. The N-terminus of LctM completely dehydrated its substrate LctA with efficiency similar to WT LctM. The N-terminus of CylM also dehydrated CylLs completely. The N-terminus of ProcM catalyzed one dehydration efficiently in ProcAs, but in the presence of the C-terminus, the dehydration reactions were complete. These results may suggest a different mode of catalysis for these LanMs. The precursor peptides of lanthipeptides are ribosomally translated and are composed of an N-terminal leader peptide and a C-terminal core peptide. Sequence alignments of class II lanthipeptide leader peptides showed that they are typically rich in aspartates and glutamates. The leader peptide is believed to be recognized by the biosynthetic enzymes and guide the process of the post-translational modifications. Moreover, it is proposed that the binding of leader peptides to the enzymes shifts the equilibrium of the inactive and active enzymes to the latter form. Efforts were made to obtain an LctA leader peptide with better binding affinity to LctM for the following reasons: 1. Increased binding affinity of LctA to LctM might result in improved catalytic efficiency, and 2. Increased binding affinity might facilitate finally obtaining a crystal structure of a LanM with the substrate bound to it. Yeast surface display was selected to engineer the LctA leader for better binding affinity to the LctM enzyme. Two rounds of sorting resulted in several mutations in the leader region, including three consistent aspartate mutations. Using fluorescence polarization assay, it was confirmed that the binding affinity of the mutant leader peptide was about 5-fold stronger than the WT leader peptide. Furthermore, LctA containing the engineered leader peptide was shown to be a more efficient substrate for LctM.
Issue Date:2015-11-17
Rights Information:Copyright 2015 Yi Yu
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12

This item appears in the following Collection(s)

Item Statistics