17. Cloning and expression of functional domains of PyrD and PyrR as a bifunctional enzyme for potential use in the biofortification of Riboflavin in plants.

Cloning and expression of functional domains of PyrD and PyrR as a bifunctional enzyme for potential use in the biofortification of Riboflavin in plants Jose Izaguirre Garcia, Lilia Saber, Sargent Briana, and Ghulam Hasnain Abstract Riboflavin (vitamin B2) is the precursor of the flavin cofactors, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The deaminase and reductase steps in riboflavin biosynthesis are catalyzed by the bifunctional enzyme RibD in Escherichia coli. Plants have two homologues of RibD, PyrD (At4g20960), and PyrR (At3g47390). The plant’s PyrD protein is known to be a degenerate deaminase-reductase in which the reductase domain has lost its critical substrate binding residues, and, thus all activity related to it. Conversely, the plant’s three-domain PyrR protein has lost its zinc-binding residues and was shown to have lost its deaminase activity. Our goal is to reconstruct the functional deaminase and reductase domains of the PyrD and PyrR proteins and make a bifunctional enzyme by fusing together the deaminase domain of PyrD (At4g20960) and the reductase domain of PyrR (At3g47390). The reconstructed gene will be tested for functional activity in a chromosomal ΔribD deletant mutant bacterial strain CmpX13 and studied in vitro. The recombinant enzyme kinetic data will be compared with the kinetic activities of PyrD and PyrR for confirmation. This study may allow us to discuss the evolutionary advantage of plants in deactivating either of these domains. Furthermore, the reconstructed deaminase-reductase bifunctional gene may be a useful gene in the biofortification study of riboflavin in plants