In the present study, we have discovered by genetic and biochemical approaches that Ferrostatin-1 in vitro xanthosine phosphorylase (xapA; also known as purine nucleoside phosphorylase II [PNP-II], EC 2.4.2.1) is also capable of converting NAM to NR in E. coli. XapA was originally identified from E. coli, and known to catalyze the reversible ribosyltransfer on purine nucleosides including xanthosine, inosine and guanosine [35–37]. Our data has not only assigned a novel function to xapA, but also uncovered a potential new route in the NAD+
salvage, in which the pathway III is extended by using NAM as an alternative precursor in xapA-possessing organisms. Results Genetic PF-01367338 molecular weight 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 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 (see Table 1, Additional file 1: Figure S1 and Additional file 2: Table S1). We first generated a mutant strain deficient in NAD+ de novo pathway (BW25113ΔnadC) that was unable to survive in the M9 minimal medium, but could restore the growth to a level comparable to the wild-type BW25113 when NA or NAM was supplied to allow NAD+ synthesized via NAD+ salvage pathway I (Figure 2 and MK-1775 mw Table 2). Table 1 Escherichia coli strains and plasmids used in this study Strains or plasmids Genotypes and comments Source or reference Strain DH5α Routine cloning host In-house collection BW25113 rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 CGSC* BW25113ΔnadC BW25113 with chromosomal nadC deletion This study BW25113ΔnadCΔpncA BW25113 with chromosomal nadC and pncA deletion This study BW25113ΔnadCΔpncAΔxapA N-acetylglucosamine-1-phosphate transferase BW25113 with chromosomal nadC, pncA, and xapA deletion This study BW25113ΔnadCΔpncAΔnadR BW25113 with chromosomal nadC, pncA, and nadR deletion This study
BW25113ΔnadCΔpncAΔxapAΔnadR BW25113 with chromosomal nadC, pncA, xapA and nadR deletion This study Plasmid pKD13 Gene knockout procedure CGSC* pKD46 Gene knockout procedure CGSC* pCP20 Gene knockout procedure CGSC* pBAD-hisA bla + In-house collection pBAD-EGFP pBAD-hisA with EGFP gene This study pBAD-xapA pBAD-hisA with xapA gene This study pET28a Kana + In-house collection pET28-xapA pET28a with xapA gene This study pEGFP-N2 Template for PCR amplification of EGFP gene In-house collection *CGSC is the E. coli Genetic Stock Center of Yale University. Figure 2 Growth of wild-type Escherichia coli (BW25113) and mutants in LB or M9 agar plates supplied with NAM or NA. Strains in area I-VI represent BW25113, BW25113ΔnadC, BW25113ΔnadCΔpncA, BW25113ΔnadCΔpncAΔxapA, BW25113ΔnadCΔpncAΔnadR and BW25113ΔnadCΔpncAΔxapAΔnadR, respectively.