It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase
Figure 3
The final reaction steps of vitamin B6 biosynthesis take place at a second active site in Pdx1.
(A) RP-HPLC elution profile of tryptic peptides of Pdx1 based on the absorbance at 215 nm, following PLP reconstitution and reduction to trap the final product. The gray line indicates the elution profile based on the fluorescence emission at 390 nm upon excitation at 320 nm. (B) MALDI-TOF/TOF analysis of the precursor ion m/z = 1758.8 [M+H]+; a predominant loss of 98 Da corresponds to loss of phosphoric acid (m/z = 1660.8 [M+H]+), while a further loss of 133 Da corresponds to loss of the pyridoxal moiety (m/z = 1527.8 [M+H]+) matching the unmodified peptide. (C) MALDI-TOF/TOF analysis of the precursor ion m/z = 1527.8 [M+H]+, yielded a fragmentation pattern matching the predicted profile for the peptide TKGEPGTGNIVEAVR (residues 148–162). Observed β (red) and γ (blue) ions are indicated.