Nitric oxide (NO), long thought of purely as a poison, experienced a renaissance in study beginning in the 1970’s leading to the discovery that NO is a crucial secondary messenger in pathways throughout the human body, including the activation of guanylate cyclase to produce cGMP and eventually the relaxation of smooth muscle in blood vessels; providing evidence that NO can play a beneficial biological role. There is also a growing field of study focused on the identification and characterization of enzymes that utilize NO as a co-substrate in the production of natural products such as the cytochrome P450s (CYPs) TxtE and RufO, found in species of Streptomycetes, that catalyze the regioselective, aromatic nitrations of L-Trp and L-tyrosine to 4-NO2-L-Trp and 3-NO2-L-tyrosine, respectively.
Historically, canonical CYPs are associated with the activation and incorporation of dioxygen (O2) into aliphatic substrates in the form of hydroxyl or epoxide groups. Work in this dissertation focuses on how TxtE diverges from canonical CYP hydroxylation activity and instead reacts with diffusible NO to catalyze the regioselective nitration of its substrate. Work in this presentation demonstrates that an intermediate (ferric-superoxo) that TxtE shares with canonical CYPs, is resistant to further reduction in TxtE. Additionally, a Thr250Ala mutant form of TxtE was generated, characterized, and found to completely lack nitration activity despite complete retention of the heme cofactor, showing that outer-sphere influences play a critical role in the tuning of TxtE towards nitration activity. A collection of data in the form of spectroscopic characterization (UV-vis absorption and EPR) of the TxtE active site, stopped-flow kinetic assays of TxtE autoxidation and reaction of the ferric-superoxo intermediate with NO, and product analysis utilizing liquid-chromatography mass-spectrometry (LC-MS) will be presented.
The collection of work presented contributes to our general understanding of how certain metalloenzymes have evolved to utilize NO in a productive fashion. Understanding of the mechanism and structure-function relationships of such enzymes will guide the engineering of such enzymes for use as biocatalysts in the regioselective nitration of industrially-relevant compounds at ambient temperature and pressure in aqueous conditions.
Dr. Jonathan Caranto (chair).
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Meeting ID: 915 1414 4300
Passcode: 751744
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