Genome Mining for Structurally Novel Class II Lanthipeptides and Probing the Conformational Dynamics of PhnZ Using Hydrogen Deuterium Exchange Mass Spectrometry PDF Download

Author: Sally Hamry
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Languages : en
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Book Description
"Nature provides a remarkable suite of natural products and biochemical catalysts (enzymes) that have proven beneficial for medicine and agriculture. This research first employs a genome mining-based approach to identify structurally novel lanthipeptide natural products. Lanthipeptides are a subfamily of genetically encoded ribosomally-synthesized and post-translationally modified peptides (RiPPs) that possess characteristic thioether bridges. The thioether bridges are installed by multifunctional enzymes (lanthipeptide synthetases) and confer biological activity to the peptide. Both the synthetases and the macrocyclized peptide products have drawn interest from the research community for the biocatalytic and biomedical properties, respectively. Using a plethora of bioinformatic tools, we have discovered several unique lanthipeptide biosynthetic gene clusters in the bacterial phylum, Actinobacteria. These include unusual glycine-rich lanthipeptides produced by the human symbiont, Rothia dentocariosa M567, as well as highly macrocyclized lanthipeptides from the soil actinomycete, Micromonospora saelicesensis DSM 44871. Preliminary structural characterization of the enzymatically modified peptides suggests that these peptides possess structurally novel sets of thioether linkages that may allow for novel biological functions. In support of this, the M. saelicesensis DSM 44871lanthipeptides were shown to stimulate plant growth. In a second research project, I investigate PhnZ, a metal ion-dependent phosphohydrolase of the HD superfamily. PhnZ is the second component of a two-enzyme oxidative pathway that works alongside PhnY to generate the nutrients inorganic phosphate and glycine. PhnZ shows extraordinary ability to perform the oxidative cleavage of a highly stable carbon phosphorous (CP)-bond using a mixed valence diiron (II/III) cofactor and O2 to generate glycine and Pi. PhnZ can potentially be exploited to break down glyphosate (Roundup) - an extensively used herbicide that has garnered controversy for contaminating unintended targets. However, the native specificity of PhnZ must be tuned to cleave the CP-bond of glyphosate. Enzyme engineering can successfully manipulate enzyme specificity, but a thorough understanding of the structure and function of PhnZ is required. X-ray crystallography has revealed that PhnZ must undergo extensive conformational changes to accomplish CP-bond cleavage. Yet, this is only a snapshot of the whole story as crystal structures alone cannot provide an accurate description of solution-phase protein structural fluctuations. Here we aim to gain a more detailed understanding of the conformational dynamics of PhnZ and the atomic-scale fluctuations that occurs over multiple timescales. A bottom-up continuous labeling hydrogen deuterium exchange mass spectrometry workflow was implemented to investigate the change in conformational dynamics of PhnZ upon substrate binding to a nonreactive substrate mimic, 1-hydroxyethylphosphonic acid"--