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The Synthesis and Enzymatic Characterization of 6-Amino-FMN with BluB; The Last Unsolved Biosynthetic Module of Vitamin B12
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The last unsolved module for the biosynthetic pathway of vitamin B12 is the biosynthesis of the lower axial ligand, dimethylbenzimidazole (DMB). Recently, the oxygen-dependent enzyme, BluB, has been shown to catalyze the oxidative fragmentation of coenzyme B2, FMN, and produces dimethylbenzimidazole (DMB), erythrose-4-phosphate, and alloxan. BluB is the first enzyme shown to utilize FMN as a substrate rather than as a coenzyme. Our goal is to elucidate the mechanism by which BluB catalyzes the oxidative fragmentation of FMN with molecular oxygen. Previous studies, done by our group, with numerous substrate analogues have shown no enzymatic activity with BluB and conclude that the active site of BluB is highly selective for the native substrate. In this study, the synthesis of 6-amino-FMN and its activity with BluB are described. LCMS analysis of the enzymatic assays confirmed the production of 4-amino-DMB. This discovery suggests that active site of BluB tolerates substrate analogues with substituents at the 6-position and additional 6-substituted substrate analogues can be designed to provide mechanistic insight.
Diaz, Daniel Jesus (2016). The Synthesis and Enzymatic Characterization of 6-Amino-FMN with BluB; The Last Unsolved Biosynthetic Module of Vitamin B12. Undergraduate Research Scholars Program. Available electronically from