UBC Theses and Dissertations
Structural and mechanistic studies on the biosynthesis of streptozocin Henderson, Alyssa
N-nitroso containing natural products, which contain the core structure −N−N=O, can act as both chemotherapeutics and as carcinogens due to their ability to alkylate DNA. While the enzymes responsible for the formation of this functional group have also been proposed as potential green alternatives to traditional synthetic methods, little is known about their mechanisms of action. One N-nitroso containing natural product, streptozocin, has been used as a chemotherapeutic since the 1960s, but the biosynthetic machinery responsible for its synthesis remained unknown until recent years. In this research, I obtained crystal structures of two enzymes involved in the biosynthesis of streptozocin: StzF, the enzyme that is believed to form the N-N bond, and StzK, an enzyme with a yet-unknown function that is proposed to ligate an N-nitroso-containing-compound to an unidentified glucosamine donor. The StzF and StzK structures were obtained at resolutions of 1.6 and 1.8 Å, respectively. StzF is a unique enzyme composed of 3 domains: an N-terminal domain, a diiron domain, and a cupin domain. An anomalous signal was observed that indicates that StzF binds iron. To explore the behavior of the metal centers of this enzyme, EPR studies were conducted that allowed for the detection of an iron-nitrosyl species that may be an intermediate in N-N bond formation. To probe the prevalence of N-nitroso compounds in the environment, bioinformatic searches were conducted. The presence of sequences encoding N-nitroso forming machinery was observed in a variety of deposited genomes, suggesting that these compounds may be isolable from the corresponding organisms.
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