- Genes specifying the thiamin monophos¬phate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for thia¬min diphosphate (ThDP) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to thia¬min di¬phos¬phokin¬ase (Tnr3) in Schizo¬sacc¬¬h¬aromyces pombe was evaluated as a candidate for these funct¬ions. Com¬par¬¬ative genomic analysis predicted a role in thiamin metabolism, not biosyn¬th¬esis, because free-standing homologues of this Nudix dom¬ain occur not only in fungi and plants, but also in proteo¬bacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosph¬ate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recomb¬¬inant Tnr3 and its Saccharo¬myces cerevisiae, Arabid¬opsis, and maize Nudix homo¬logues lacked thiamin monophosphate phos¬phatase activity but were active against ThDP, and up to 60-fold more active against diphos¬ph¬ates of the toxic thiamin degradation pro¬ducts oxy- and oxo¬thi¬amin. Deleti¬ng the S. cere¬visiae Nudix gene (YJR142W) lower¬ed oxythiamin resistance, over-expressing it rais¬ed resist¬ance, and express¬ing its plant or bacterial counterparts restored resist-ance to the YJR142W deletant. By converting the di¬phos¬phates of damaged forms of thiamin to monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincor¬p¬or¬ation of damaged diphosphates into ThDP-de¬pend¬ent enzymes, and the resulting toxicity.