Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function.

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as and epimers. The epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B-related genes in bacteria, and (iii) epimerase and B-related genes are coexpressed in yeast and The predicted second function was explored in , whose epimerase and dehydratase are fused and encoded by The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in YjeF). Changing this residue to alanine cut epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B.