Structural studies of flavoprotein inhibitors and inactivators
[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI--COLUMBIA AT REQUEST OF AUTHOR.] Flavoenzymes have a versatile capacity to catalyze a wide range of reactions, using their flavin cofactor. In this thesis, work on two flavoenzymes is presented. The bifunctional enzyme SmPutA, from Sinorhizobium meliloti is composed of the proline dehydrogenase (PRODH) and glutamate semialdehyde dehydrogenase (GSALDH) domains. SmPutA was used as a surrogate for the human enzymes, to identify proline analogs that bind the active sites of PRODH and GSALDH, which are valuable chemical probes to study the roles of proline metabolism in cancer. Binders of the enzymes were searched for in crystallo resulting in the serendipitous identification of two distinct new mechanisms of covalent inactivation of PRODH. The first, presented in Chapter 1, thiazolidine-2-carboxylate (T2C) was characterized in the active site of PRODH. T2C was found in the active site of PRODH covalently bonded to the N5 of the flavin. Although the inactivation by T2C was slow, it led us to the discovery of two related inactivators of PRODH: tetrahydrothiophene-2-carboxylate (C2C) and 1,3-dithiolane-2-carboxylate (D2C), presented in Chapter 2. The inactivators D2C and C2C differed from T2C by only one atom, but surprisingly they inactivated the PRODH enzyme through an entirely different mechanism, which was triggered by light. D2C and C2C are decarboxylated in the active site and become bound to the N5 of the flavin via their C2 atoms. A mechanism starting with the light-generated triplet excited state of FAD is proposed. Additionally, non-covalent binders of GSALDH have been structurally characterized in the active site of GSALDH, in Chapter 3. Isomers of proline and hydroxyproline were able to bind the active site. Although the binding affinity is low, the contacts revealed in the structures could be leveraged when designing more potent inhibitors of GSALDH. Finally, Chapter 4 presents a series of crystal structures of the ornithine hydroxylase SidA, which reveal a dynamic flavin and active site.
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