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

Biosci Rep. 2018 May 8;38(3):BSR20180223. doi: 10.1042/BSR20180223. Print 2018 Jun 29.

Abstract

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 R and S epimers. The S 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 B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro 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 in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF 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 B6.

Keywords: AIBP; NAD(P)H hydrates; NAD(P)HX; NAXE; Vitamin B6; protein moonlighting.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Arabidopsis / enzymology
  • Catalytic Domain
  • Conserved Sequence / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Humans
  • Mutation
  • NAD
  • NADP
  • Pyridoxal Phosphate / chemistry
  • Pyridoxal Phosphate / metabolism*
  • Racemases and Epimerases / chemistry*
  • Racemases and Epimerases / deficiency
  • Racemases and Epimerases / genetics
  • Saccharomyces cerevisiae / enzymology
  • Stereoisomerism
  • Vitamin B 6 / chemistry*
  • Vitamin B 6 / genetics

Substances

  • NAD
  • NADP
  • Pyridoxal Phosphate
  • Vitamin B 6
  • NAXE protein, human
  • Racemases and Epimerases