Date of Award

May 2023

Degree Type


Degree Name

Doctor of Philosophy



First Advisor

Nicholas R Silvaggi

Committee Members

Graham Moran, Alan Schwabacher, Jarett Wilcoxen, Arsenio Pacheco


Pyridoxal-5’-Phosphate (PLP)-dependent enzymes are ubiquitous throughout nature with over 140 unique activities characterized. This diverse reactivity has been attributed to PLP’s ability to act as an “electron sink” during catalysis. In recent years, a novel oxidase activity was characterized with the discovery of MppP, an enzyme in the biosynthetic pathway of mannopeptimycin, that catalyzes the first step in the production of the non-proteinogenic amino acid L-enduracididine. MppP catalyzes the four-electron oxidation of L-arginine to 4-hydroxy-(2,3)-dehydroarginine, using only the PLP cofactor and molecular oxygen, with no assistance from metals or other organic cofactors. Shortly after MppP was reported, Ind4 was characterized. This enzyme is part of the biosynthetic pathway for indolmycin, another antibiotic. Ind4 is also able to catalyze the four-electron oxidation of L-arginine, but instead of producing the hydroxylated product, a desaturation occurs, creating (2,3),(4,5)-didehydroarginine. Thus, two classes of L-arginine oxidases have been found: the MppP-like hydroxylases and the Ind4-like desaturases. Despite having different activities, the overall structures and the active site residues are all conserved. This new class of PLP-dependent enzymes offers not only the chance to further characterize a novel activity of a ubiquitous cofactor but also can provide a unique insight into how enzymes have evolved fine control over their cofactor’s reactivity. To this end, multiple desaturases were characterized. Crystal structures of Pel4, Plu4, and Bcer4 were determined. Pel4 and Plu4 structures initially were missing electron density for the PLP cofactor, making active site comparisons between them and hydroxylases impossible. Through a series of experiments, crystallization conditions were found such that ordered the cofactor sufficiently to observe the electron density. Comparisons of the active sites of Plu4 and SwMppP provide little insight into how the enzymes are able to catalyze different reactions, though some subtle differences in active site residues provide some hints.

Pre-steady state kinetic experiments were conducted with Plu4. These data were fitted to a model using global fitting by simulation. One issue with the mechanistic model of SwMppP is the parameter-to-data ratio. Three of the four observable signals are due to species occurring before reaction with dioxygen. This means that the latter half of the mechanism is not well constrained by the data. The desaturated product made by Plu4 provides extra observables. Using these new observables, the latter half of the mechanism of Plu4 is better constrained. These stopped flow data of Plu4 now allow for kinetic characterization of Plu4 and comparison to the previously characterized SwMppP. Together with the sequence and structural data, these data allow predictions of important residues that were investigated using mutagenesis. Two mutants of Plu4, D216N and R121S, were characterized. These mutations help provide a basis of how oxygen activation occurs and how L-arginine oxidases have evolved to catalyze two different reactions.

Available for download on Thursday, May 30, 2024

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