Vitamin B?? or Cobalamin is structurally the most complex of all the vitamins. The entire biosynthetic pathway of cobalamin is well studied except the formation of the lower ligand known as dimethylbenzimidazole (DMB). In aerobic pathway, BluB catalyzes the formation of DMB using reduced flavin mononucleotide (FMNH?) as its substrate leading to the formation of DMB and erythrose4-phosphate as the end products. It is a remarkable transformation, wherein, C2 of DMB is derived from C1' of the ribose sugar chain of FMN. However, the fate of the ring C of FMN moiety as well as the overall mechanism of this unique reaction is still unknown. Thus, identification of the final unknown product and a detailed mechanistic study of dimethylbenzimidazole formation is the main focus of this work. In this dissertation, we have successfully identified alloxan as the end product derived from the third ring of FMN. Water and molecular oxygen have been shown to be the two sources of oxygen atom incorporation in alloxan based on O-18 labeling studies. A key intermediate in our mechanistic proposal has been successfully trapped in the form of six different shunt products using water, bisulfite and hydride as nucleophiles. Trapping of this intermediate helps us establish that the C-C bond cleavage occurs first forming erythrose-4-phosphate followed by the release of alloxan. Asp-32 has been shown to play an important role in stabilizing this intermediate. Based on stereochemical studies, we have shown that the pro-R hydrogen is selectively abstracted from the C1' of the ribityl chain of the substrate. This result helps us establish that the final oxidation step involved in DMB formation is indeed catalyzed by the enzyme. Formation of a new shunt product was observed on using 8-substituted flavins as substrate analogs for the BluB catalyzed reaction. Characterization of this shunt product provides evidence for the initial fragmentation of the peroxyflavin intermediate involved in the DMB biosynthesis. All these above observations are consistent with our current mechanistic proposal for the DMB formation. Thus, we have finally unraveled the long unsolved mystery in the Vitamin B?? biosynthesis.