Ergothioneine and ovothiol A are naturally occurring thiol-histidine derivatives. Both of them are suggested to be beneficial to human health. Ergothioneine has anti-inflammation and anti-oxidative properties and ovothiol has anti-proliferative activities. Recently, ergothioneine has been suggested to be also linked to lifespan longevity. For these reasons, there is a need to investigate the mechanisms of ergothioneine and ovothiol biosynthesis is appealing. My thesis work has addressed this gap in knowledge, focusing on the mechanistic investigations of two C-S bond formation enzymes: EanB in anaerobic ergothioneine biosynthesis, and OvoA in ovothiol biosynthesis.
Chapter 1 provides an overview of sulfur-containing metabolites, including the metabolism, potential biological functions, and biosynthesis of several key sulfur containing natural products.
Chapter 2 contains my initial investigations into EanB catalysis, namely the original sulfur source for this enzyme. We demonstrated that the polysulfide (HSSnSR) is the direct sulfur source in EanB catalysis. With the discovery of the unique sulfur source, we then probed how EanB uses polysulfide for catalysis. A few reaction intermediate states were successfully characterized by X-ray crystallography and the proposed reaction mechanisms were further evaluated by QM/MM calculation.
In Chapter 3, we evaluated the involvement of a proposed carbene intermediate involved in EanB catalysis by the deuterium exchange experiments with hercynine. In addition, using 3,5-difluoro-tyrosine containing EanB produced through amber suppressor method, we have also kinetically characterize the deuterium-exchange reaction.
Chapter 4 reports the biochemical characterization of an OvoA homolog, OvoAMtht, from a mesophilic organism. OvoAMtht has dual activities: sulfoxide synthase and cysteine dioxygenase. In addition, I have demonstrated that both substrates and the active site iron’s secondary coordination shell residues exert exquisite control to OvoAMtht dual activities, which makes OvoAMtht an excellent system for future structure-function relationship studies for this class of enzymes.
In summary, my thesis has laid the foundation for future detailed mechanistic investigations of the C-S bond formation reactions in both anaerobic ergothioneine biosynthetic and ovothiol aerobic biosynthetic pathways.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/44913 |
| Date | 21 July 2022 |
| Creators | Cheng, Ronghai |
| Contributors | Liu, Pinghua |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
Page generated in 0.0042 seconds