Structural Insights Into An Aromatic Nitration Catalyzed Enzyme In Pharmaceutical Industry

Aromatic nitration is the most common process for the production and preparation of aromatic nitro compounds which involving a large number of important chemical raw materials, particularly in the field of fine chemicals as represented in drug synthesis, structure pinpoint selective nitration has been an urgent requirement, such as the famous "lifestyle" drug sildenafil core intermediate source that is achieved through aromatic nitration. The optimization of aromatic nitration reaction process has gone through a long time to phase out the "mixed acid" program which must have concentrated sulfuric acid participated, and then developed a series of liquid or gas phase nitration reaction with catalyst as the core, but still never been able to get rid of nitric acid using, leaving the byproducts control, the reaction environment-friendly optimization and the atom economy improvement as the urgent problems. With the rise of life science advances and synthetic biology, green enzyme catalysis to achieve important industrial reaction becomes more and more attractive and promising.Cytochrome P450(CYPs) are heme-dependent enzymes can catalyze a wide variety of oxidation reaction through oxygen activatetion. As a ubiquitous superfamily they are involved in processes ranging from lipid and drug metabolism in mammals to triterpenoid modification in plants and biosynthesis and biodegradation in microbes. Their unique ability to catalyze a diverse array of oxidative transformations of a wide variety of compounds has made them a sustained academic topic of research since their discovery in1958. CYPs not only in the organic body act as important catabolic role, they also have a pivotal role in the synthesis of numerous natural products. Although some time that people are concerned about less often, and even to some extent, can be said to be ignored, but in fact this area is the "center stage" allow CYPs really shine. Which involves a large number of chemical reactions is very fascinating, and their recognition of the complex substrate is really stunning. In this regard, CYPs performance and did not like its original metabolic roles "inclusive" and is usually completed only responsible for a specific step in the synthesis of specific synthetic pathway, which means that they can be imagined to have a very specific substrate object recognition.CYP TxtE which catalyzed the directly nitration of L-tryptophan indole ring is an important enzyme on the Streptomyces scabies phytotoxic Thaxtomin A biosynthetic pathway, and it is the only cytochrome P450family member recent identified directly catalyze aromatic nitration with nitric oxide as raw material. This thesis choose TxtE as the research object, trying to explore the biological structure of this enzyme and to identify the mechanism of substrate recognition and catalyzation, laying the foundation for further protein engineering to achieve the possible molecular transformation and industrial applications.In this thesis, following heterologous recombinant expression, purification, crystallization, the three-dimensional crystal structure of TxtE is successfully resolved with homology modeling and molecular replacement, and on the basis of the work carried out to a certain extent its substrate recognition feature explored. The structure of TxtE clearly shows a substrate entering channel as well as a unique electronic delivery channel, subsequently the substrate recognition and binding patern in the activity center is preliminarily determined. Discussion on TxtE structure and substrate recognition characteristics may contribute to future green nitrification process, but also helps us understand the molecular mechanism of the CYP family.A briefly describetion about structural study of BUNV nucleoprotein and natural product oriented high-throughput drug screening assays setting up works the author participated is also included in the appendix.