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

Abstract

Background: 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. 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. Results: Additional 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. Conclusions: Both 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. © 2014 Dong et al.; licensee BioMed Central Ltd.