Abstract Characterizing the roles of biotin biosynthetic enzymes of Bacillus subtilis and Mesorhizobium loti Miglena Manandhar and John Cronan Biotin is an important enzyme cofactor that carries carboxyl group for carboxylation, decarboxylation and transcarboxylation reactions. It is a vitamin that is required by all life forms and is synthesized by majority of bacteria, archaea, fungi and plants. Previous studies of biotin biosynthetic pathway have shown that the second half of this pathway, from pimelate thioester to biotin, is well conserved in most of the organisms, and has been well characterized. However, the first half of the pathway that generates the pimelate thioester intermediate varies in terms of involvement of specific genes in different organisms and remains to be fully characterized. BioZ and BioW are two novel enzymes that have been postulated to be responsible for the generation of the pimelate thioester intermediate in the biotin biosynthesis in M. loti and B. subtilis, respectively. The amino acid sequence of BioZ has been found to be similar to a condensing enzyme of the fatty acid biosynthesis, β-ketoacyl-acyl carrier protein synthase III (FabH). This led us to hypothesize that BioZ might function as a condensing enzyme in the initial steps of biotin biosynthesis in M. loti. BioW has been proposed to be a pimeloyl-CoenzymeA (CoA) synthetase catalyzing the reaction of converting pimelic acid to pimeloyl-CoA in B. subtilis. The goal of this project is to determine the specific function of these enzymes in the biotin biosynthetic pathway by carrying out genetic as well as biochemical experiments.