Biochemical evidence on the synthesis of NR from NAM catalyzed by E. coli xapA as determined by HPLC-ESI-MS/MS. A) Selected-ion monitoring (SIM) chromatogram at m/z 254.3-255.3 Da of NR converted from NAM by recombinant xapA. B) Positive ESI-MS/MS spectrum of the NR peak produced by xapA and eluted from HPLC showing an ion fragmentation pattern characteristic to NR, including two major peaks representing NR and the NAM moiety with m/z at 255 and 123, respectively. C) SIM chromatogram of NR converted from NAM by CIAP as positive control. D) Positive ESI-MS/MS spectrum of the NR peak produced by CIAP and eluted from HPLC. The retention time and MS/MS spectra were identical to those produced by standard NR control, confirming the synthesis of NR from NAM by xapA. See Figure 1 for abbreviations.