Capturing the time resolved structures of enzymes undergoing catalysis is a grand challenge of structural biology. While this approach has been well developed for light activated reactions, the general approach of mixing enzyme crystals with activating reagents and substrates has only been applied to a handful of cases to date. In this project, serial crystallography (combining data from many thousands of small crystals to form a complete dataset) will be used to obtain structures at ambient (i.e. non-cryogenic) temperatures using microcrystals for two different multifunctional enzymes. Data will be obtained using synchrotron and X-Ray free electron laser sources and X-Ray emission and other spectroscopies will be used to identify reaction intermediate states. Enzyme reactions will be initiated by mixing of crystals with either hydrogen peroxide or molecular oxygen and reactions followed at microseconds to seconds time points. The heme enzymes to be studied are (i) the remarkable multifunctional globin dehaloperoxidase (DHP) that is able to catalyse oxidase, peroxidase, oxygenase and peroxygenase reactivities; (ii) catalase peroxidase (KatG). DHP appears able to tune reactivity based on the substrates encountered although the mechanisms for this remain unclear in the absence of time resolved structures of reactions. KatG activates the pro-drug isonicotinic acid hydrazide (INH) a first line of treatment for tuberculosis for over 60 years. Despite the success of INH, the activation mechanism remains elusive. Neither enzyme has been studied in a time resolved manner.
Outstanding training opportunities are available within this studentship, with the opportunity to work at world leading X-Ray sources and engage with a broad collaborative team.
Applications for this project have now closed.
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