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

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 homologs of RibD, PyrD (At4g20960) and PyrR (At3g47390). The plant PyrD protein is known to be a degenerate deaminase-reductase in which the reductase domain has lost critical substrate binding residues and hence activity. The plant three-domain PyrR protein has lost the zinc-binding residues and is recently shown lacks deaminase activity. Interestingly, both the PyrD and PyrR are multi-domain proteins in which reductase domain and deaminase domains deactivated respectively. We have created a chimeric gene in which the functional domains of PyrD and PyrR are fused. To test the activity of the plants reconstructed bifunctional deaminase/reductase enzyme we have created a riboflavin auxotrophic E. coli RibD deletant mutant (ΔribD::Kan), which also carriers a riboflavin transporter (RibM) from C. glutamicum. After confirming in vivo activity of reconstructed deaminase/reductase gene we will express the gene in E. coli and purify the recombinant protein to study the enzyme activity in vitro. The recombinant enzyme kinetic data will be compared with the kinetic activities of PyrD and PyrR. This study may allow us to discuss the evolutionary advantage of deactivating the reductase domain in PyrD and the deaminase domain in PyrR in plants. The reconstructed deaminase-reductase bifunctional gene may be a useful gene in the biofortification study of riboflavin in plants.