Fig. 6: Functional validation of the critical role of (R/Q)xW motif of cyclic di-pyrimidine-synthesizing CdnE.
A Schematic representation of the mutational strategy for re-wiring the substrate and product specificity. ITC analysis of the interaction between (B) non-hydrolysable GMPcPP and EfCdnER279A and (C) non-hydrolysable AMPcPP and EfCdnEW281A. The determined dissociation constants (KD, M) were indicated. The experiments were repeated at least twice. LC-MS/MS analysis of the products synthesized by incubating (D) EfCdnER279A with 1:1 UTP/GTP mixture and (E) EfCdnEW281A with 1:1 UTP/ATP mixture. In (D), a peak of m/z 649.01 and its fragmented ions was found to be eluted at 6.1 min having the same elution time of cyclic UMP-GMP. In (E), a peak of m/z 633.03 and its fragmented ions was found to be eluted at 6.1 min having the same elution time of cyclic UMP-AMP. F The cartoon model (based on ClCdnE-UTP complex) showing the substrate and product preference of cyclic di-pyrimidine-synthesizing CdnE. The conserved (R/Q)xW motif in donor pocket and conserved asparagine residue (N) plus two donor UTP and acceptor UTP were shown in spheres. The substrates can be UTP or CTP. The products were identified to be cUU, cUC and cCC.
