Functional Characterization of Metalloenzymes in Lumichrome Catabolism and Chuangxinmycin Biosynthesis
Abstract
Cofactors play vital roles in augmenting the limited functionality available on proteins for catalysis. Little is known about cofactor breakdown in contrast to the extensive literature on cofactor biosynthesis and enzymology. Our lab discovered strains capable of degrading riboflavin and lumichrome. We identified the lumichrome catabolic gene cluster in Pimelobacter simplex and reconstituted most of the biodegradation reactions.
I report the reconstitution of 8 enzymes, including a cytochrome P450 monooxygenase, a molybdenum enzyme, a rieske dioxygenase, and a catechol dioxygenase, in the lumichrome catabolic pathway. Cytochrome P450 (quinoxaline monooxygenase) hydroxylates the C7 position of lumichrome. Nature does this to increase the solubility of lumichrome for biodegradation. The pyrimidine ring of 7-carboxylumichrome is then hydrolyzed by amidohydrolases. Rieske dioxygenase (quinoxaline dioxygenase) carries out cis-dihydroxylation of dihydroxyquinoxaline-carboxylic acid to open the quinoxaline heterocycle. Amidocatechol dioxygenase catalyzes ring cleavage with oxygen insertion to form as yet unidentified ring-opened product.
CxnD, cytochrome P450 monooxygenase, catalyzes carbon-sulfur bond formation to form demethyl-chuangxinmycin. It was recently biochemically and structurally characterized by Shi et al., but the mechanism of C-S bond formation is poorly understood. We proposed two plausible mechanisms for the formation of aryl carbon and sulfur bond via i) aryl radical anion intermediate ii) (2-3)-epoxyindole intermediate. We tested the proposal by feeding several substrate analogs to CxnD. Bromo-substituted substrate analogs ruled out the carbon-sulfur bond formation via aryl radical anion intermediate. Intriguingly, indole-3-propanoic acid, as a mechanistic probe, releases five products that support the mechanism involving (2-3)-epoxyindole intermediate for C-S bond formation.
Subject
Metalloenzymeslumichrome
riboflavin
rieske dioxygenase
catechol dioxygenase
molybdenum enzyme
cytochrome P450
chuangxinmycin
Citation
Naseem, Saad (2022). Functional Characterization of Metalloenzymes in Lumichrome Catabolism and Chuangxinmycin Biosynthesis. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /197092.