Little is known about cofactor catabolism in the literature. Pyridoxamine, thiamin, heme and nicotinic acid degradation pathways are the only well-characterized catabolic pathways. In 2012 the Begley lab isolated Microbacterium maritypicum G10 strain, from DSM Nutritional Products, Germany, which can degrade riboflavin (Vitamin B2). Subsequent screening with the cosmid library made from the strain has led to the identification of the riboflavin catabolic gene cluster. One of the gene cluster products - the enzyme riboflavin lyase (RcaE), is a flavoenzyme that catalyzes the oxidative degradation of riboflavin to lumichrome and ribose. The enzymes encoded by the other genes of the cluster have been predicted by bioinformatics to include a flavokinase (RcaA), a flavin reductase (RcaB) and a ribokinase (RcaD). These enzymes have been reconstituted individually. The main focus of this work is the detailed mechanistic analysis of riboflavin lyase (RcaE). Reconstitution of the enzyme RcaE requires reduced FMN as the cofactor with riboflavin and oxygen acting as substrates. Thus, RcaE serves as the first example where the reduced cofactor FMN catalyzes the breakdown of a second flavin molecule, riboflavin. In this degradation, the participation of a superoxide radical, derived from reduced FMN reacting with molecular oxygen at the enzyme active site, has been established by direct superoxide incorporation experiments. RcaE is a unique enzyme that uptakes superoxide directly and utilizes it to degrade the substrate. Till date, there is no report in the literature of an enzyme where a superoxide radical abstracts a hydrogen atom from the substrate. Based on our results with the radical trap cyclopropylmethylflavin, we propose that the superoxide radical mediates a one electron oxidation of the substrate riboflavin, by a hydrogen atom abstraction from the C1? position of riboflavin to form riboflavin-C1?-radical and hydrogen peroxide. The riboflavin-C1?-radical in a second one electron oxidation gets converted to riboflavin-C1?-imine. Based on the LCMS experiments showing ^18O incorporation into the ribose from ^18O2, we further propose that the resulting imine recombines with the hydrogen peroxide at the active site to give riboflavin-C1?-hydroperoxide. Finally, through reduction to riboflavin-C1?-hydroxide and subsequent hydrolysis, the riboflavin-C1?-hydroperoxide converts to lumichrome and ribose.
