Biosynthesis of vitamin B6: direct identification of the product of the PdxA-catalyzed oxidation of 4-hydroxy-l-threonine-4-phosphate using electrospray ionization mass spectrometry

PdxA (E.C. 1.1.1.262) catalyzes a key step in the biosynthesis of vitamin B6: the nicotinamide-dependent oxidation of 4-hydroxy-l-threonine-4-phosphate (HTP) to a product tentatively identified as 3-amino-1-hydroxyacetone 1-phosphate (AHAP). To date, the evidence for the formation of AHAP, while self-consistent, has been largely circumstantial, and does not exclude the possibility that the actual product of the enzyme-catalyzed oxidation of HTP might be 2-amino-3-oxo-4-hydroxybutyric acid 4-phosphate which could undergo rapid, non-enzyme-catalyzed decarboxylation once released from the protein. Use of negative ion electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometric analysis (MS-MS) confirms that AHAP is the product of the PdxA-catalyzed reaction.