New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide

Wei-Ren Dong,Guan Zhu,Li-Xin Xiang,Jian-Zhong Shao,Cen-Cen Sun,Shi-Hua Hu

doi:10.1186/1471-2180-14-29

Abstract

BackgroundIn an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD+de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD+ salvage pathway from nicotinamide.ResultsAdditional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation.ConclusionsBoth the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD+ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD+ salvage pathway might be significant in some bacteria lacking NAD+de novo and NAD+ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD+. However, this speculation needs to be experimentally tested.

Highlights

In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+ de novo and salvage pathways
NAD+ can be synthesized de novo from simple amino acid precursors such as tryptophan or aspartate, or converted from intermediates such as nicotinamide (NAM), nicotinic acid (NA) or nicotinamide riboside (NR) via salvage pathways, which are designated as salvage pathways I (i.e., (NAM→) NA → NaMN [nicotinic acid mononucleotide] → deNAD [deamino-NAD] → NAD+), II (i.e., NAM → NMN [nicotinamide mononucleotide] → NAD+), and III (i.e., NR → NMN → NAD+), respectively
Genetic disruption of NAD+ de novo biosynthesis and NAD+ salvage pathway I in Escherichia coli In an effort to uncover the new function of E. coli xanthosine phophorylase (xapA) in NAD+ salvage pathway from nicotinamide, we produced a set of gene knockout mutants deficient in previously defined NAD+ synthetic pathways, including NAD+ de novo and NAD+ salvage pathways I and III for genetic investigation purpose

Summary

Introduction

In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+ de novo and salvage pathways. The mutant deficient in NAD+ de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. Nicotinamide adenine dinucleotide (NAD+) and NAD+ phosphate (NADP+) are two of the most important coenzymes in cells. They act as either electron donors or electron acceptors in more than 300 enzymatically catalyzed oxidoreductions [1,2].

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