| dc.description.abstract | The present work describes studies aimed at characterizing enzymes involved in bacterial metabolic pathways using a variety of biochemical methods, analytical techniques and structural studies. The first study explains the structural and biochemical characterization of a C-glycosidase involved in pseudouridine catabolic pathway. Our studies suggested that its mechanism is significantly different from the previously reported glycosidases. The second study describes the discovery and mechanistic characterization of a radical SAM enzyme involved in a new menaquinone biosynthetic pathway. This enzyme represents a unique reaction motif in radical SAM enzymology.
Pseudouridine (Ψ), the most abundant modification in RNA, is synthesized using Ψ synthase. Recently, a pathway for the degradation of Ψ was described in Escherichia coli. In this pathway, Ψ is first converted to Ψ 5′-monophosphate (ΨMP) by Ψ kinase and then ΨMP is degraded by ΨMP glycosidase to uracil and ribose 5- phosphate. The structural studies on the ΨMP glycosidase and its mutant (K166A) suggested that the reaction utilizes a Lys166-substrate adduct during catalysis. Biochemical studies on ΨMP glycosidase further confirmed the existence of a lysine adduct and allowed us to identify roles for specific active site residues. ΨMP glycosidase catalyzes the cleavage of the C−C glycosidic bond through a novel ribose ring-opening mechanism. This is the first mechanistically characterized C-glycosidase.
Menaquinone (MK, vitamin K2) is a lipid soluble molecule that participates in the bacterial electron transport chain. In mammalian cells, MK functions as an essential vitamin for the activation of various proteins involved in blood clotting and bone metabolism. Recently, a new pathway for the biosynthesis of this cofactor was discovered in Streptomyces coelicolor A3(2) in which chorismate is converted to aminofutalosine in a reaction catalyzed by MqnA and an unidentified enzyme. Here, we reconstitute the biosynthesis of aminofutalosine and demonstrate that the missing enzyme (aminofutalosine synthase, MqnE) is a radical SAM enzyme that catalyzes the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl) oxy] benzoic acid. This is a new reaction type in the radical SAM superfamily. The substrate analogs based mechanistic investigation suggested that MqnE catalyzes a unique radical rearrangement reaction, unprecedented in biological chemistry. | en |
