Date of Completion

2019

Document Type

Honors College Thesis

Department

Chemistry

Thesis Type

College of Arts and Science Honors, Honors College

First Advisor

Matthew Liptak

Keywords

drug development, protein structure, mutagenesis, porphyrin complex, drug-resistance

Abstract

Staphylococcus aureus is one of the most common causes of infection and has been rapidly developing drug resistance, making the discovery of a new drug target imperative. This project analyzed the interaction between heme and IsdG, a heme degrading enzyme involved in the spread of S. aureus, to gain further insight into the mechanism of heme degradation. Iron serves as an essential nutrient for S. aureus, and IsdG is the primary enzyme involved in iron acquisition from heme. IsdG has a catalytic pocket that contains tryptophan at residue 67 that is expected to be involved in heme ruffling. The W67F variant of IsdG formed with site-directed mutagenesis was expected to ruffle and degrade heme to a lesser extent being that Trp is replaced with a less bulky amino acid, Phenylalanine (Phe, F). The variant was characterized using Electronic Absorption (Abs), Ultraviolet Circular Dichroism (UV CD), and Magnetic Circular Dichroism (MCD) spectroscopies to analyze changes to the geometric and electronic structure resulting from the substitution. The CD spectra confirmed similar geometric structures of W67F and wild-type (WT) IsdG. The activity of W67F differed from that of WT IsdG, indicating that Trp67 is involved in the enzyme’s activity and promotes IsdG turnover. The Abs spectra showed a blue-shift in the Q and Soret bands of W67F IsdG indicating reduced heme ruffling and that Trp67 was involved in heme ruffling. The increased intensity of the negative-component of the Soret band in the MCD spectrum of W67F IsdG was indicative of a 2Eg electronic ground state, consistent with reduced heme ruffling. Being that Trp67 is an active site residue specific to the IsdG family of non-canonical heme oxygenases (HOs), this information regarding the influence of Trp67 on heme ruffling and IsdG activity is crucial to the development of a selective inhibitor of IsdG in the presence of canonical HOs.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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