Studies on the Flavin-mediated Cysteine Salvage Pathway, Structural Enzymology of Riboflavin Lyase and Mechanistic Investigation of the Radical S-adenosyl-L-methionine-mediated Tryptophan Lyase

This dissertation discusses mechanistic and structural studies of enzymes with unique features. The studies are focused on: (i) mechanistic investigation of a novel cysteine salvage pathway that involves a flavoenzyme-mediated Pummerer-type rearrangement; (ii) structural enzymology of the superoxide radical-mediated catabolism of riboflavin; and (iii) mechanistic studies on a radical-mediated fragmentation-recombination reaction catalyzed by Tryptophan Lyase (NosL). Amino acid cysteine and its metabolites play a vital role in all forms of life. Recently, a pathway was identified in Bacillus subtilis that salvages cysteine from Ssubstituted cysteines. We have reconstituted all five enzymes involved in this transformation. Detailed biochemical studies were performed on a unique Pummerer-type rearrangement catalyzed by a flavin monooxygenase, CmoJ. Though the pathway shows broad substrate specificity, we have identified the physiological substrates of the enzymes. This study provides insights into a novel pathway that extracts sulfur from the organosulfur molecules in the largely unexplored world of rhizosphere microbiome. In contrast to the biosynthesis of cofactors, their catabolism is scantly described in literature. The first riboflavin catabolic pathway was recently identified in Begley lab in 2013. Mechanistic investigation using biochemical studies revealed that flavin mononucleotide (FMN) dependent Riboflavin Lyase (RcaE) utilizes a superoxide radical to catabolize riboflavin. Crystal structure of RcaE was solved at 1.75 Å, bound with cofactor FMN and substrate riboflavin. Orientations of these ligands in the active site demonstrate that stacking of the riboflavin on top of FMN prevents the formation of typically observed C₄a-flavin(hydro)peroxide. This structural study provides insights into a new class of flavin-dependent enzymes. Tryptophan Lyase (NosL) is a radical SAM enzyme that catalyzes the formation of 3-methyl-2-indolic acid from L-tryptophan. As suggested by the recent crystal structure of NosL, we have obtained biochemical evidence for H-atom abstraction from the amino group of L-tryptophan. Further experiments demonstrate that the subsequent β-scission involves Cα-C(O) cleavage. A mechanistic proposal is discussed that incorporates all latest discoveries, including the newly identified byproducts of the NosL catalyzed reaction. Additional studies with substrate analogs as well as mutants demonstrate a cornucopia of reactions with the 5′-deoxyadenosyl radical, which initiates chemistry in all radical SAM enzymes