Thiamin (Vitamin B1) is made from a coupling a thiazole and a pyrimidine unit, which are assembled separately. Studies have shown that the biosyntheses of thiazole and pyrimidine are different in prokaryotes versus eukaryotes. Understanding of thiamin biosynthesis is still incomplete and a lot of new discoveries relating to the enzymes in its biosynthesis have been explored in depth revealing new mechanisms and enzymology. In prokaryotes, five different enzymes are known to be directly involved in thiamin thiazole biosynthesis. The in vitro reconstitution of this enzymatic pathway has been achieved and detailed insights have been obtained, however, the very small quantity of product produced in this in vitro reconstitution prevented direct characterization of its structure. We were able to study the last few steps on the prokaryotic thiamin thiazole pathway in greater detail, and elucidate the structure of the final product of the thiazole synthase to be the thiazole tautomer phosphate. We were also able to assign function to a gene involved in aromatization of the unstable thiazole tautomer phosphate to the thiazole carboxylate phosphate. We also knew that a single gene product ThiC converts amino-imidazole ribonucleotide, an intermediate in the purine nucleotide biosynthesis, to HMP, using a complex rearrangement reaction. This enzyme had been very difficult to isolate and study biochemically because it was air-sensitive and its cofactors were unknown. We recently were able to show that it was a [4Fe-4S] cluster containing enzyme, and belonged to the radical SAM family. The 4Fe-4S cluster binding motif (CX2-CX4-C) of ThiC is different from the motif (CX3-CX2-C) conventionally used by the other established members of this family. With the pure protein with a well-reconstituted Fe-S cluster, we were able to achieve remarkable enhancement in activity in vitro, in a defined biochemical system. True products of this reaction were thus identified to be HMP-phosphate and 5'-deoxyadenosine. We were also able to establish the fate of all the C atoms of the substrate, and have other insights into the mechanism of this complex enzyme with regard to the unprecedented rearrangement it brings about. Further mechanistic characterization of the remarkable rearrangement reaction catalyzed by ThiC is in progress.