2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases of fungi and archaea

The pathway of riboflavin (vitamin B2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. Specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). We compare 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases from the yeast Candida glabrata, the archaeaon Methanocaldococcus jannaschii and the eubacterium Aquifex aeolicus. All three enzymes convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, as shown by 13C-NMR spectroscopy using 2,1',2',3',4',5'-13C6]2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate as substrate. The beta anomer was found to be the authentic substrate, and the alpha anomer could serve as substrate subsequent to spontaneous anomerisation. The M. jannaschii and C. glabrata enzymes were shown to be A-type reductases catalysing the transfer of deuterium from the 4(R) position of NADPH to the 1' (S) position of the substrate. These results are in agreement with the known three-dimensional structure of the M. jannaschii enzyme.