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

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.

Cysteine-independent Catalase-like Activity of Vertebrate Peroxiredoxin 1 (Prx1)

“…Prx1 open reading frames (ORFs) of T. nigroviridis (TnPrx1) and H. sapiens (HsPrx1) were amplified by RT-PCR from mRNA isolated from pufferfish kidney and HeLa cells (corresponding to GenBank accession numbers DQ003333 and NM_001202431, respectively) and cloned into the pET28a bacterial expression vector containing a His 6 tag at the N terminus as described (19). TnPrx1 mutants were generated by site-directed mutagenesis by replacing all three Cys residues (i.e.…”

“…Table 1 for details on the genotypes of constructs). Recombinant Prx1 proteins were expressed in Escherichia coli and purified from the soluble fractions by nickel-nitrilotriacetic acid-agarose bead-based chromatography and eluted with elution buffer containing 250 mM imidazole or as specified (19). Purified Prx1 proteins were subjected to SDS-PAGE analysis and stained with Coomassie Brilliant Blue.…”

Peroxiredoxin 1 (Prx1) is a dual-function enzyme by possessing Cys-independent catalase-like activity

“…Fusion proteins were purified from the soluble fractions by Ni-TNA agarose bead- or amylose resin-based affinity chromatography and eluted with elution buffer containing 250 mM imidazole (for His-tagged proteins) or 10 mM maltose (for MBP-tagged proteins) in accordance with the manufacturer's instructions (Novagen (pET28 system) and NEB (pMAL system)), or as specified [19,20]. Purified Prx1 proteins were subjected to SDS-PAGE analysis and stained with Coomassie Brilliant Blue.…”

Revisiting Caffeine Biosynthesis – Speculations about the Proximate Source of its Purine Ring

“…For reasons of simplicity, possible salvage cycles [45] were omitted, but may eventually explain the asymmetric alkaloid formation (Figure 1). Therefore, I feel the urge to point to a recent work showing that a xanthosine phosphorylase is capable of using nicotinamide as a substrate for nicotinamide riboside formation [46].…”