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 to lack deaminase activity. Interestingly, both the PyrD and PyrR are multi-domain proteins in which their reductase domains and deaminase domains are deactivated, respectively. We have reconstructed the functional deaminase and reductase domains of PyrD and PyrR and made a bifunctional plant enzyme. For the reconstruction of the bifunctional enzyme, we fused the DNA sequences corresponding to PyrD deaminase domain (At4g20960) and PyrR reductase domain (At3g47390). To test the functional activity of the reconstructed putative plant bifunctional enzyme, we created a chromosomal knockout of RibD (ΔribD deletant mutant) in E. coli strain CmpX13. The reconstructed fusion gene of PyrD and PyrR was cloned in pETDuet1 plasmid. Complementation assay showed that the fusion gene of PyrD and PyrR was able to revert the auxotrophy of Riboflavin E. coli ΔribD::Kan strain. The reconstructed deaminase-reductase bifunctional gene may be a beneficial gene in the biofortification study of riboflavin in plants.
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