The Structural And Functional Research Of Human Phosphoribosyl Pyrophosphate Synthase PRS1and Its Related Proteins

Phosphoribosyl pyrophosphate synthase is responsible for the synthesis of PRPP and AMP using ATP and R5P as substrates. Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated with multiple diseases such as hyperuricemia and gout, Charcot-Marie-Tooth disease-5(CMTX5), Arts syndrome, X-linked nonsyndromic sensorineural deafness (DFN2) and even cancers because of single base mutations. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism.Here, we solved the wild type structure of PRS1and6of its pathogenetic mutant structures using X-Ray crystallography. We also reconstructed the3D cryo-EM structure of the PRS1apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we proved the hexamer is the functional unit, which also provided the quaternary structure interaction interfaces. With our combined findings based on mutagenesis, biochemical assays and sequence analysis, we reveal conserved substrates and inhibitor binding motifs. Based on these findings, we propose a possible dynamic catalytic mechanism and PRPP release tunnel. The possible mechanism has broad implications for understanding all pathogenic mutants and provides a framework for future therapy or drug design investigations.The D52H missense mutation of PRS1will lead to a conspicuous PRPP content elevation in the erythrocyte of patients and finally induce hyperuricemia and serious gout. In this study, the enzyme activity analysis indicated that D52H-mutant possessed similar catalytic activity to the wild type PRS1, and the2.27A resolution D52H-Mutant crystal structure revealed that the stable interaction network surrounding the52position of PRS1would be completely destroyed by the substitution of histidine. Combined enzyme activity assays, ITC experiments, and structural analysis, we proved that these interaction variations would further influence the conformation of ADP binding pocket of D52H-mutant and reduced the inhibitor sensitivity of PRS1in patient’s body. It might be the D52H pathogenetic mechanism, which also confirmed the viewpoint in the last paper.Many proteins including PAP39, PAP41, PRS2, PRS3and KIX domain of p300are reported to interact with PRS1. It has been reported that PAP39might be a negative regulator of PRS1, however, the influence of the interaction between PRS1and p300KIX domain is unknown. Here, we first confirmed the in vitro interaction between PAP39and PRS1as1:1(molar ratio) by conducting an N-his6-tagged PAP39pull down experiment. Then, we assayed the enzyme activity. PAP39alone displayed no activity, however, when it was added to equal molar amounts of PRS1it displayed a2-fold increased kcat compared with PRS1alone. This result implied that PAP39is an activator of PRS1. Our result was contradictory to an earlier report from1994; however, we stand behind our more rigorous experiment. In addition, we also confirmed the in vitro interaction between p300KIX domain and PRS1as1:1(molar ratio) by conducting a C-his6-tagged PRS1pull down experiment.