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

Author:

Niehaus Thomas D.1,Elbadawi-Sidhu Mona2,Huang Lili3,Prunetti Laurence4,Gregory Jesse F.3,de Crécy-Lagard Valérie4,Fiehn Oliver2,Hanson Andrew D.1

Affiliation:

1. Horticultural Sciences Department, University of Florida, Gainesville, FL, U.S.A.

2. NIH West Coast Metabolomics Center, Genome Center, University of California Davis, Davis, CA, U.S.A.

3. Food Science and Human Nutrition Department, University of Florida, Gainesville, FL, U.S.A.

4. Microbiology and Cell Science Department, University of Florida, Gainesville, FL, U.S.A.

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry,Biophysics

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