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

Thomas D Niehaus,Laurence Prunetti,Lili Huang,Jesse F Gregory,Valérie De Crécy-Lagard,Mona Elbadawi-Sidhu,Andrew D Hanson,Oliver Fiehn

doi:10.1042/bsr20180223

Thomas D Niehaus, Laurence Prunetti + Show 6 more

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https://doi.org/10.1042/bsr20180223

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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.

Highlights

Damaging enzymatic and chemical side reactions in all organisms convert NADH and NADPH to their hydrates, NADHX and NADPHX, which exist as R and S epimers [1,2]
The functions of the epimerase and the dehydratase are supported by biochemical evidence from mammals, yeast, Escherichia coli, and plants [5,6,7], and that of the dehydratase is supported by genetic and metabolomic evidence from Arabidopsis [6]
As most epimerase genes are fused to dehydratase genes, the clustering data do not show whether the pyridoxal -phosphate (PLP) connection is with the epimerase, the dehydratase, or both

Summary

Introduction

Damaging enzymatic and chemical side reactions in all organisms convert NADH and NADPH to their hydrates, NADHX and NADPHX, which exist as R and S epimers [1,2]. The hydrates, which inhibit various dehydrogenases [3,4], are reconverted to NAD(P)H by the sequential actions of NADP(H)X epimerase (EC 5.1.99.6) and NAD(P)HX dehydratase (EC 4.2.1.93) [5,6,7] (Figure 1A). Both enzymes occur in all domains of life [5]. The dehydratase is specific for the S form of NAD(P)HX [1] and the epimerase facilitates conversion of the R form to the S form used by the dehydratase [5,6,7]. The functions of the epimerase and the dehydratase are supported by biochemical evidence from mammals, yeast, Escherichia coli, and plants [5,6,7], and that of the dehydratase is supported by genetic and metabolomic evidence from Arabidopsis [6]

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