Probing the roles of Orf20 from methylobacterium extorquens AM1in the dephospho-tetrahydromethanopterin biosynthesis and T110 in the nucleotidyl substrate specificity of ADP-glucose pyrophosphorylase from agrobacterium tumefaciens

Dephospho-tetrahydromethanopterin (dH4MPT) from Methylobacteriurn extorquens AM1 and ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Agrobacterium turnefaciens play a role in carbon assimilation to give rise to new materials and products. Our current hypothesis is that Orf20 catalyzes the second step in dH4MPT biosynthesis and that a conserved threonine (T110), a member of a conserved "GTAD" motif located in the vicinity of the nucleotide substrate binding pocket confers purine substrate specificity in ADP-Glc PPase. We used a combined bioinformatics and biochemical approach to investigate the roles that Orf20 and T110 play in their respective pathways.;To elucidate the specific role of Orf20, a Malachite Green assay was developed to detect pyrophosphate released during enzyme catalysis. Computational modeling was used to gain a better understanding of the structure of Orf20, potential ligand binding sites, and a possible mechanism of catalysis. Computational mutagenesis was used to investigate the role that T110 plays in the nucleotidyl substrate specificity of ADP-Glc PPase. Under possible optimal conditions for the assay (30°C, pH 7.8), the specific activity of Orf20 was estimated to be 0.012 micromoles min-1mg-1. The T110A and T110V ADP-Glc PPase variants were generated in silica. Simulated ATP and UTP docking was used to explore the purine/pyrimidine bias of ADP-glucose pyrophosphorylase. Predicted binding energies of UTP and ATP indicate that the T11 OA variant binds UTP slightly better than ATP as compared to the T110V variant. This work provides the first biochemical evidence that Orf24 may catalyze the second step of dH4MPT biosynthesis and contributes to our fundamental understanding of microbiological sources of greenhouse gases. This work also provides the first bioinformatics evidence that the nucleotidyl substrate specificity of ADP-Glc PPase may be enhanced to accept alternative pyrimidine nucleotides to produce designer sugar-nucleotides that serve a variety of biological and therapeutic purposes.