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Title:Metabolite biosynthetic enzymes: catalysis, substrate specificity and protein engineering
Author(s):Estrada, Paola
Director of Research:Nair, Satish K
Doctoral Committee Chair(s):Nair, Satish K
Doctoral Committee Member(s):van der Donk, Wilfred A; Cronan, John E; Procko, Erik
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Protein Crystallography
natural products
Abstract:Small molecules are an important part of how bacteria survive, primary metabolites are involved in growth and development while secondary metabolites can function as signaling molecules, scavengers or chemical weapons. These small molecules have a myriad of diverse chemical structures that grant them biomedical relevance; however, their biosynthetic origin is not always known. Characterization of the enzymes that catalyze the biosynthesis of these small molecules can provide us with targets in our fight against microbes, scaffolds for their use as bioengineered tools or serve as a means to identify new biologically relevant molecules. Here I describe my efforts to (i) characterize some of the enzymes responsible for the biosynthesis of primary and secondary metabolites and (ii) engineer these enzymes to expand the structural diversity of their products. Biotin is a small molecule that plays an important role in primary metabolism as the required cofactor in carboxylation and decarboxylation reactions. One of the steps in biotin biosynthesis in some bacilli strains is the conjugation of CoA to the seven-carbon dicarboxylate pimelate, a biotin precursor. The reaction takes place in two steps, first is the activation of the carboxylate through an adenylate intermediate followed by a thioesterification reaction to co-A resulting in pimeloyl-CoA. In the biotin biosynthetic pathway, the protein in charge of these two reactions is BioW, an adenylating enzyme that represents a new protein fold within the superfamily of adenylating enzymes. Here I show substrate-bound structures that identified the enzyme active site and elucidated the mechanistic strategy for pimelate-CoA production. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. BioW has also been shown to hydrolyze adenylates of noncognate substrates and data shown here indicates that his activity can be abolished by mutation of a single residue. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products produced by secondary metabolism. A precursor peptide is translated by the ribosome and tailoring enzymes perform multiple post-translation modifications on said peptide transforming it into the final natural product. Notably, the tailoring enzymes are encoded in the same gene cluster as the precursor peptide. In the second chapter, I show how mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of cyanobactins, a class of RiPPs made by cyanobacteria. Most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on acceptor substrates. However, in the case of the natural product piricyclamide 70005E1, an O-geranylation, not prenylation, is present on a Tyr residue. PirF is the enzyme that carries out the reaction utilizing a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the isoprene-binding pocket, relative to the C5 utilizing enzymes. Mutating this single amino acid is enough to completely switch the donor specificity from a C5 to a C10. I also show that these enzymes may be used for the chemospecific attachment of C5 or C10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. Lastly, I show work trying to characterize enzymes that are part of a putative lanthipeptide biosynthetic gene cluster from Lachnospiraceae bacterium C6A11, the final product of which is not yet known. This cluster contains diverse modifying enzymes that have not been seen to be encoded together in any previously characterized cluster. One of these enzymes is LahD1, a protein belonging to the YcaO family of enzymes. The YcaO family of enzymes have been shown to catalyze the formation of azoles, thioamides and amidines but the role of LahD1 is still unknown. In efforts to identify its activity I was able to get the crystal structure of LahD1 and see the location of its Iron-Sulfur cluster, a co-factor never seen before in YcaOs. Another enzyme part of the lah cluster characterized here is the LahSB, a substrate tolerant methyltransferase that modifies the C-terminal end of peptides.
Issue Date:2019-12-02
Rights Information:Copyright 2019 Paola Estrada
Date Available in IDEALS:2020-03-02
Date Deposited:2019-12

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