Qualitative and Quantitative NAD+ Metabolomics Lead to Discovery of Multiple Functional Nicotinate N-Glycosyltransferase in Arabidopsis.

Lingyun Liu,Guodong Wang,Guosheng Li,Fengxia Zhang

doi:10.3389/fpls.2019.01164

Abstract

The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is a conserved biochemical pathway in land plants. We previously demonstrated that various NA conjugations (mainly methylation and glycosylation) shared the NA detoxification function in all tested plants. It remains unclear whether other NA conjugates with low abundance exist in plants. In this study, we discovered at least two additional NA N-glycosides in Arabidopsis, which was tentatively elucidated as nicotinate N-pentoside (NaNP) and NA N-rhamoside (NaNRha), using liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS) with precursor ion-scanning (PreIS). We further quantitatively profile the NAD+-related metabolites in 24 tissues of Arabidopsis. Biochemical assays of UGT76C family revealed that UGT76C5 (encoded by At5g05890, previously identified as NaNGT) was a multiple functional nicotinate N-glycosyltransferase, with high preference to UDP-xylose and UDP-arabinose. The deficiency of NaNP and NaNRha in ugt76c5 mutant suggested that UGT76C5 is responsible for biosynthesis of NaNP and NaNRha in planta. We also identify one amino acid difference in PSPG (plant secondary product glycosyltransferase) motif is responsible for the divergence of NaNGT (UGT76C4) and UGT76C5. Taken together, our study not only identifies a novel nicotinate N-glycosyltransferase but also paves the way for investigations of the in planta physiological functions of various NA conjugations.

Highlights

It has long been known that NAD is a coenzyme that serves as an electron carrier in hundreds of redox reactions
NA detoxification conferred by nicotinate N-methyltransferase (NaNMT, EC 2.1.1.7, encoded by At3g53140) might have facilitated the retention of the Preiss-Handler pathway in land plants (Li et al, 2017)
Novel NA Conjugates Detected in Arabidopsis thaliana

Summary

INTRODUCTION

It has long been known that NAD is a coenzyme that serves as an electron carrier in hundreds of redox reactions. The Preiss-Handler pathway, shared by land plants and most bacteria, starts with nicotinate (NA), which generated by nicotinamidase (NIC1, EC 3.5.1.19, encoded by At2g22570) from nicotinamide (NAM). Multiple Functional Nicotinate N-Glycosyltransferase (Figure 1) (Preiss and Handler, 1958a, Preiss and Handler, 1958b; Wang and Pichersky, 2007). NA detoxification conferred by nicotinate N-methyltransferase (NaNMT, EC 2.1.1.7, encoded by At3g53140) might have facilitated the retention of the Preiss-Handler pathway in land plants (Li et al, 2017). The reversible methylation at carboxyl group of NA had been characterized as long-distance transport of NAD precursors in Arabidopsis thaliana (Wu et al, 2018). One amino acid in PSPG (plant secondary product glycosyltransferase) motif is responsible for the biochemical difference of UGT76C4 (NaNGT) and UGT76C5

MATERIALS AND METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT