17. Side directed mutagenesis studies of PyrD and PyrR genes involved in riboflavin biosynthesis

Abstract

Riboflavin (vitamin B2) is the precursor of the flavin cofactors, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Plants and many microorganisms, synthesized riboflavin but animals must obtain it from dietary sources (Power, 2003). 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. Although the missing zinc finger binding residues of reductase domain in PyrD, and changes in critical substrate binding residues in the reductase domain of PyrR are shown using sequence alignments but have not studied experimentally that the loss of function of deaminase and reductase activity in PyrR and PyrD, respectively, are due to changes in these amino acid sequences.

In this project, we are using side directed mutagenesis approach to change the critical substrate-binding residues of the deaminase domain of the PyrR gene and the addition of missing Zinc finger binding residues in the PyrD gene. We have used the E. coli RibD gene as a benchmark. To test the mutated PyrD and PyrR we have created a riboflavin auxotrophic E. coli RibD deletant mutant (ΔribD::Kan), which also carriers a riboflavin transporter (RibM) from C. glutamicum. Cloning of mutated PyrD and PyrR are in progress